TUGAS MATA KULIAH

FISIOLOGI DAN TEKNOLOGI REPRODUKSI VETERINER

(Inseminasi Buatan)

Inseminasi Buatan pada Kuda

Oleh
Gede Wiyasa Ardy Nugraha
1709511059
Kelas B

FAKULTAS KEDOKTERAN HEWAN

UNIVERSITAS UDAYANA
TAHUN 2020
 KATA PENGANTAR

Puji syukur saya panjatkan ke hadirat Tuhan Yang Maha Esa, karena dengan
rahmat dan hidayah-Nya saya dapat menyelesaiakan paper yang berjudul
“Inseminasi Buatan pada Kuda”.
Meskipun banyak rintangan dan hambatan yang saya alami dalam
proses pengerjaannya, tapi saya berhasil menyelesaikannya dengan baik. Adapun
paper ini dibuat guna memenuhi tugas mata kuliah Fisiologi dan Teknologi
Reproduksi Veteriner di Fakultas Kedokteran Hewan Universitas Udayana.
Saya sebagai penulis mengucapkan rasa berterimakasih yang sebesar-
besarnya kepada semua pihak yang telah membantu saya sehingga dapat
menyelesaikan paper ini tepat waktu. Saya yakin paper ini masih jauh dari nilai
kesempurnaan. Oleh karena itu, kritik dan saran yang bersifat membangun sangat
diharapkan oleh penulis demi menjadikan paper ini bisa lebih baik lagi.
Semoga paper " Inseminasi Buatan pada Kuda " memberikan informasi yang
berguna bagi masyarakat serta bermanfaat untuk pengembangan wawasan dan
peningkatan ilmu pengetahuan bagi kita semua.

Denpasar, 26 Maret 2020

Penulis

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 DAFTAR ISI

Cover
Kata Pengantar .................................................................................................... i
Daftar Isi............................................................................................................. ii
Daftar Gambar ................................................................................................... iii
Daftar Tabel ...................................................................................................... iv
Daftar Lampiran ................................................................................................. v
Bab I Pendahuluan
1.1 Latar Belakang ............................................................................................ 1
1.2 Rumusan Masalah ....................................................................................... 2
1.3 Tujuan Penulisan ......................................................................................... 2
Bab II Tinjauan Pustaka
2.1 Tujuan Inseminasi Buatan pada Kuda ........................................................ 3
2.2 Koleksi Semen pada Kuda .......................................................................... 4
2.3 Evaluasi Semen pada Kuda ......................................................................... 9
2.4 Penyimpanan dan Penggunaan Semen pada Kuda ................................... 18
2.5 Pengenceran Semen pada Kuda ................................................................ 19
2.6 Volume Inseminasi Buatan pada Kuda ..................................................... 19
2.7 Teknik Inseminasi Buatan pada Kuda ...................................................... 20
Bab III Penutup
3.1 Simpulan ................................................................................................... 21
3.2 Saran ......................................................................................................... 21
Daftar Pustaka .................................................................................................. 22

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 DAFTAR GAMBAR

Gambar 1. Representasi diagram dari vagina buatan equine (AV) .................... 5

Gambar 2. (A) Cambridge AV; (B) Missouri AV; (C) Nishikawa AV; (D)
Hannover AV ...................................................................................................... 5
Gambar 3. Untuk melindungi vagina buatan dari sinar ultraviolet dan untuk
membantu mempertahankan suhu yang dibutuhkan, semuanya bisa ditutup
dalam selubung pelindung................................................................................... 6
Gambar 4. Dummy mare .................................................................................... 7
Gambar 5. Beberapa contoh kelainan yang umum yang mungkin terlihat saat
memeriksa sperma untuk morfologi................................................................. 16

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 DAFTAR TABEL

Tabel 1. Contoh ekstender digunakan untuk evaluasi semen ........................... 11

Tabel 2. Kriteria untuk menilai pergerakan sperma .......................................... 13

iv
 DAFTAR LAMPIRAN

Lampiran 1. Viability and fertility of cooled equine semen diluted with skimmed
milk or glycine egg yolk-based extenders.
Lampiran 2. Effects of different artificial insemination techniques and sperm
doses on fertility of normal mares and mares with abnormal reproductive history.
Lampiran 3. Factors Influencing the Popularity of Artificial Insemination of
Mares in Europe.

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 BAB I PENDAHULUAN

1.1. Latar Belakang

Kuda telah didomestikasi lebih dari 6.000 tahun yang lalu di daerah stepa
yang sekarang dikenal dengan daerah Rusia Selatan dan Ukraina. Sejak itu
kuda mempunyai banyak manfaat yang berhubungan dengan manusia.
Penduduk asli Indonesia telah beternak kuda sebelum kedatangan Eropa. Kuda
hidup pada saat itu di alam bebas dan sangat tergantung pada kebaikan alam
sehingga kuda yang dipelihara memiliki kualitas rendah. Kedatangan Portugis
dan Belanda ke Indonesia memiliki andil memperbaiki ras kuda lokal,
termasuk memperbaiki cara beternak seperti cara pemberian makan yang baik,
perawatan kuda, serta petunujuk-petunjuk lain yang berhubungan dengan kuda.
Ternak kuda termasuk komoditas ternak yang ada di Indonesia dan belum
mendapat perhatian yang proporsional baik oleh pemerintah maupun oleh
masyarakat. Keberadaan ternak kuda dinilai cukup strategis karena fungsinya
sebagai ternak kerja (salah satunya adalah kuda penarik andong) dan memiliki
nilai estetika yang menarik. Penelitian tentang ternak kuda sampai saat ini
belum banyak dilakukan oleh pakar di bidang peternakan bahkan publikasi
ilmiah tentang ternak kuda di Indonesia sangat langka, pembahasan dan diskusi
mengenai perkembanganya hampir tidak mendapat perhatian.
Dewasa ini, terjadi penurunan jumlah kelahiran kuda dikarenakan masih
kurangnya tempat pembudidayaan kuda yang memenuhi standar, serta masih
kurangnya pengetahuan para peternak kuda tentang siklus reproduksi kuda. Hal
tesebut menyebabkan para peternak kuda tidak dapt menentukan masa kawin
atau musim kawin yang tepat bagi kuda sehingga jumlah kelahiran kuda tidak
mencapai titik optimal.
Penelitian khusus ke dalam inseminasi buatan (IB) pada equine terbatas,
karena sejumlah masyarakat peternak masih tidak akan menerima untuk
keturunan yang dikandung dengan cara ini. Yang paling penting dari semua ini
di Inggris adalah Asosiasi Peternak Unggul, yang pada giliran, menyediakan
sejumlah besar dana baik secara langsung maupun tidak langsung untuk
penelitian kuda, terutama yang berkaitan dengan reproduksi. Sebagian besar

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 masyarakat peternak lain di Inggris dan di seluruh dunia sekarang menerima
keturunan IB, tetapi banyak yang menetapkan peraturan ketat, seperti batas
jumlah anak kuda yang dapat didaftarkan per kuda jantan per tahun. Di Eropa,
Australia, Cina, Afrika Selatan dan Amerika Serikat, IB kuda sekarang tersebar
luas dalam penggunaannya. Penentangan historis terhadap IB membatasi
penelitian dan menghambat pengembangan teknik. Karena itu memiliki
beberapa cara yang harus ditempuh sebelum mencapai kecanggihan IB ternak.

1.2. Rumusan Masalah

Berdasarkan latar belakang yang telah dibuat dapat dibuat rumusan
masalah yaitu
1.2.1 Apa tujuan melakukan inseminasi buatan pada kuda?
1.2.2 Bagaimana cara mengkoleksi semen pada kuda?
1.2.3 Bagaimana cara evaluasi semen pada kuda?
1.2.4 Bagaimana cara penyimpanan dan penggunaan semen pada kuda?
1.2.5 Bagaimana cara pengenceran semen pada kuda?
1.2.6 Berapa volume inseminasi buatan pada kuda?
1.2.7 Bagaiamana teknik melakukan inseminasi pada kuda?

1.3. Tujuan Penulisan

Berdasarkan rumusan masalah diatas dapat dibuat tujuan penulisan yaitu
1.3.1 Untuk mengetahui tujuan melakukan inseminasi buatan pada kuda.
1.3.2 Untuk mengetahui cara mengkoleksi semen pada kuda.
1.3.3 Untuk mengetahui cara evaluasi semen pada kuda.
1.3.4 Untuk mengetahui cara penyimpanan dan penggunaan semen pada kuda.
1.3.5 Untuk mengetahui cara pengenceran semen pada kuda.
1.3.6 Untuk mengetahui volume inseminasi buatan pada kuda.
1.3.7 Untuk mengetahui teknik melakukan inseminasi pada kuda.

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 BAB II TINJAUAN PUSTAKA

2.1. Tujuan Inseminasi Buatan pada Kuda

Ada berbagai alasan mengapa IB pada kuda dipraktekkan. Beberapa di
antaranya tergantung dan dibatasi oleh peraturan yang ditetapkan oleh negara
dan masyarakat peternak yang terlibat. Alasan untuk menggunakan IB
meliputi:
1. Penghapusan batasan geografis.
2. Meminimalkan transfer penyakit, baik kelamin maupun sistemik, dengan
menghilangkan kontak langsung antara kuda betina dan kuda jantan.
Semen juga dapat diobati dengan ekstender yang mengandung antibiotik
untuk meminimalkan kandungan bakteri dan karenanya mengurangi
jumlah organisme yang berpotensi patogen. Karena itu, semen semacam
itu berguna untuk kuda betina yang memiliki kerentanan lebih tinggi
terhadap infeksi rahim.
3. Pengurangan risiko cedera baik untuk handler dan kuda dengan
menghilangkan kontak langsung antara kuda Betina dan kuda jantan.
Risiko ini semakin berkurang jika kuda jantan dapat didorong untuk
menaiki dummy kuda betina.
4. Meningkatkan jumlah kuda betina yang dapat diinseminasi per ejakulasi.
5. Peningkatan stok asli melalui impor semen.
6. Pengembangan bank gen untuk reintroduksi materi genetik di masa depan.
7. Pembiakan kuda betina yang sulit seperti mereka yang memiliki kelainan
fisik, terutama disebabkan oleh kecelakaan, infeksi, konformasi perineum
yang buruk, masalah psikologis, dll. Namun, perhatian harus diberikan
untuk memastikan bahwa masalah tersebut tidak dapat diwariskan.
8. Pembiakan dari kuda jantan yang sulit - mereka yang memiliki masalah
fisik, cedera, infeksi, karakteristik semen yang tidak memadai, masalah
psikologis, dll. Seperti dengan kuda betina harus diambil untuk
memastikan bahwa masalah seperti itu tidak diwariskan.
9. Pengurangan biaya tenaga kerja.
10. Semen sexing.

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 Beberapa kekhawatiran tentang penggunaan IB juga telah dipahami,
termasuk pengurangan kumpulan genetik, penekanan berlebihan pada strain
'modern', keterampilan teknis yang diperlukan dan risiko infeksi jika skrining
yang memadai tidak dilakukan.

2.2. Koleksi Semen pada Kuda

Pengumpulan semen dapat dilakukan dengan menggunakan salah satu dari
beberapa metode. Metode termudah adalah pengumpulan sampel turun. Tetes
semen dikumpulkan dari kuda jantan setelah pemisahan dari kuda betina ke
dalam toples steril. Metode ini tidak dapat diandalkan, kualitas sampel sangat
bervariasi dan sebagian besar sampel tertinggal dalam kuda betina. Sampel
sering mengandung konsentrasi sperma yang rendah dan organisme patogen
yang relatif tinggi. Semen juga dapat diambil dari vagina anterior segera
setelah kawin. Namun, semen yang terkumpul sudah bersentuhan dengan asam
dan karenanya sekresi spermisida ditemukan di dalam vagina. Sperma juga
dapat terkontaminasi oleh organisme patogen. Tidak satu pun dari metode
pengumpulan ini memungkinkan penilaian terhadap total volume semen yang
diproduksi.
Kondom telah dikembangkan untuk digunakan pada kuda. Ini dapat
bekerja dengan sangat baik, tetapi memiliki kecenderungan untuk meledak atau
menjadi copot, sehingga seluruh sampel hilang.
Akhirnya, metode pengumpulan semen yang terbaik dan paling umum
digunakan adalah vagina buatan (AV). AV pertama dikembangkan untuk
digunakan pada kuda di Rusia pada awal abad ke-20. Pengembangan AV
selanjutnya adalah untuk digunakan pada ternak. Berbagai model, termasuk
Cambridge, Colorado, Missouri, Nishikawa dan Hannover, tersedia, tetapi
semuanya didasarkan pada prinsip yang sama. Ini menyediakan lumen hangat
dan steril, dikelilingi oleh selubung air, di bawah tekanan, dengan pengumpul
di bagian akhir, dalam upaya untuk meniru vagina alami.

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 Gambar 1. Representasi diagram dari vagina buatan equine (AV)

Gambar 2. (A) Cambridge AV; (B) Missouri AV; (C) Nishikawa AV; (D)
Hannover AV
Sebagian besar AV terdiri dari selubung luar yang kokoh dengan dua
lapisan karet, bagian luar dan bagian dalam. Lapisan luar dan selubung
membentuk selubung, di mana air hangat atau udara lewat melalui keran atau
katup. Suhu air di dalam AV harus sedikit di atas suhu tubuh, pada 44-48°C.
Jumlah air yang digunakan harus memadai untuk memastikan bahwa tekanan
di dalam lumen AV meniru sedekat mungkin tekanan terhadap insersi penis di
dalam vagina alami. Beberapa model (Missouri) memungkinkan tekanan
lumen ditingkatkan dengan memompa selubung air, karena udara lebih ringan
dari air, ini meminimalkan bobot akhir AV. Lapisan dalam AV sering

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 dilindungi oleh liner sekali pakai tambahan, sehingga memastikan sterilitas.
Liner sekali pakai, atau dalam beberapa kasus liner itu sendiri, terhubung ke
pengumpul. Sebelum digunakan, liner ini dilumasi dengan pelumas obstetrik
steril untuk membantu kuda jantan. Yang terpenting adalah pengumpul, serta
lumen AV, sekitar 44 ° C selama pengumpulan, untuk mencegah cold shock.
Isolasi dan perlindungan dari sinar ultraviolet dapat disediakan dengan
melampirkan seluruh AV dan penampung di dalam selubung pelindung. AV
yang dirakit penuh sebelum digunakan memiliki panjang 50 cm dan beratnya
mencapai 10 kg.

Gambar 3. Untuk melindungi vagina buatan dari sinar ultraviolet dan untuk
membantu mempertahankan suhu yang dibutuhkan, semuanya bisa ditutup dalam
selubung pelindung.
Sebagaimana dibahas, sperma rentan terhadap sinar matahari dan
perubahan suhu. Jika suhu AV atau pengumpul terlalu dingin, sperma akan
menderita dari cold shock dan mati. Jika suhu AV terlalu panas, ada efek buruk
yang serupa pada sperma. Selain itu, ada risiko mencegah kuda jantan
menggunakan AV, dan juga mungkin melakukan layanan alami. Kuda jantan
tampaknya kurang sensitif terhadap suhu dibandingkan dengan hewan ternak
lainnya, tetapi suhu di atas 48 ° C harus dihindari.
Kuda jantan dapat dilatih relatif mudah untuk menggunakan AV. Pelatihan
awal menggunakan kuda betina, disiapkan seperti penutup normal, untuk

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mendorong kawin dan ejakulasi ke dalam AV. Namun demikian, penggunaan
kuda betina estrus memiliki risiko, termasuk kecelakaan yang tidak disengaja.
Karena itu banyak kuda jantan dilatih untuk menaiki dummy kuda betina.

Gambar 4. Dummy mare

Sebagian besar kuda jantan cukup senang menggunakan dummy seperti
itu, terutama jika kuda betina estrus ada di sekitarnya. Beberapa kuda jantan
tidak begitu tajam, biasanya sebagai akibat dari libido rendah, konsekuensi
yang mungkin terjadi pada kuda jantan yang terlambat bekerja, pensiun sebagai
kuda kinerja, atau manajemen IB yang salah di masa lalu.
Kuda jantan ini mungkin membutuhkan rangsangan ekstra dari jump mare.
Kuda betina ini mungkin merupakan nymphomaniac yang terjadi secara alami
dan berada dalam keadaan estrus berkelanjutan karena ketidakseimbangan
hormon atau dia dapat diobati dengan estradiol-17β untuk menginduksi estrus
tanpa ovulasi. Namun, tidak semua kuda betina nymphomaniac cocok karena
kondisi ini dapat mengakibatkan perilaku yang tidak terduga.
Kuda jantan disiapkan untuk pengumpulan semen dengan cara yang sama
seperti yang dia lakukan secara alami. Kekang penutup harus digunakan dan
semen dapat dikumpulkan di penutup normal atau di area IB khusus. Jika jump

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mare akan digunakan, ia kembali dipersiapkan untuk layanan alami, termasuk
swab jika terjadi kecelakaan. Dia harus bersikap tenang. Ini sangat penting
selama pelatihan kuda jantan muda.
Sangat penting bahwa semua peralatan ada dan dalam urutan yang
memuaskan dan pada suhu yang tepat untuk pengumpulan dan penanganan
selanjutnya sebelum kuda jantan dibawa masuk untuk pengumpulan. Setiap
orang yang terlibat harus tahu apa yang diharapkan darinya. Pengumpulan
semen selalu membawa risiko, karena sifat kuda jantan yang tidak dapat
diprediksi, terutama saat covering. Seperti biasa dengan penutup tangan, semua
handler disarankan untuk mengenakan topi keras, terutama pengumpul semen.
Diperlukan hingga tiga handler jika jump mare akan digunakan, satu untuk
memegang kuda jantan, satu untuk mengambil semen, dan satu untuk
memegang jump mare. Semua handler harus berdiri di sisi kuda jantan yang
sama. Ini memastikan bahwa, jika terjadi kecelakaan, kuda jantan dapat ditarik
oleh handlernya, dan meminimalkan kemungkinan pengumpul semen
ditendang. Sisi yang digunakan tidak mempengaruhi sampel yang
dikumpulkan, tetapi, setelah kuda jantan terbiasa dengan semen yang
dikumpulkan dari satu sisi, yang terbaik adalah mencoba dan menempel pada
sisi itu di masa depan.
Kuda jantan diizinkan untuk dinaiki dan pengumpul mengalihkan penis,
ketika ereksi, ke arah AV. Kuda jantan harus diizinkan untuk mendapatkan
intromisi dan memasukkan AV atas kehendaknya sendiri dan tidak memiliki
AV yang dipaksakan padanya. AV dapat distabilkan dengan ditahan pada
bagian belakang kuda betina, jika ada, atau bagian belakang dummy.
Terjadinya ejakulasi dicatat, seperti pada alami, oleh pembesaran ekor atau
dengan perasaan kontraksi uretra dan lewatnya semen di sepanjang sisi ventral
penis.
Setelah pengumpulan, pengumpul harus dikeluarkan dengan hati-hati dari
AV dan semen dievaluasi sesegera mungkin. Jika tidak mungkin untuk
melakukan penilaian semen segera, itu dapat diperpanjang dan disimpan pada
suhu 4-5°C hingga 24 jam tanpa pengurangan yang cukup dalam kelayakannya,
dan evaluasi semen yang cukup akurat masih dapat diperoleh.

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2.3. Evaluasi Semen pada Kuda
Sebelum evaluasi, fraksi gel semen harus dihilangkan. Fraksi gel ini adalah
sekresi setelah dan memiliki konsentrasi sperma yang sangat rendah, yang
selalu mati. Fraksi gel semen dapat dihilangkan dengan beberapa metode:
aspirasi hati-hati dengan jarum suntik steril; filtrasi melalui kain kasa atau
dengan sistem filtrasi sebaris yang dimasukkan ke dalam AV; atau decanting
hati-hati. Filtrasi adalah metode paling populer yang digunakan saat ini dan
juga memastikan debris dihilangkan.
Sangat penting bahwa, setiap saat, termasuk selama penanganan dan
evaluasi, semen dijaga agar tetap hangat, pada suhu 38 ° C (sedikit lebih rendah
dari suhu yang sebenarnya dikumpulkan). Sperma sangat rentan terhadap cold
shock dan, jika ada instrumen, slide, tahapan mikroskop, dll, tidak dipersiapkan
sebelumnya, maka hasil yang diperoleh akan menyesatkan. Semen dapat
dievaluasi dalam beberapa kategori dan tidak semua evaluasi melibatkan
semua penilaian yang dirinci di bawah ini. Ini akan bervariasi sesuai dengan
apa yang diharapkan untuk dicapai oleh penilaian dan alasan pelaksanaannya.
Dianjurkan agar semua penilaian dilakukan sebelum kuda jantan memasuki
program IB untuk pertama kalinya, dan sebaiknya mengulanginya di awal
setiap musim. Penampilan, motilitas, konsentrasi, dan kemungkinan morfologi
harus dinilai secara ideal pada setiap sampel yang dikumpulkan.
Ketika menilai potensi reproduksi kuda jantan, yang terbaik adalah
mengumpulkan lebih dari satu sampel untuk penilaian. Dua sampel yang
mungkin direkomendasikan untuk pengumpulan semen untuk evaluasi: dua
ejakulasi diambil 1 jam terpisah, diikuti 3 hari kemudian oleh satu ejakulasi
tunggal untuk evaluasi; atau dua ejakulasi dikumpulkan 1 jam terpisah, diikuti
dengan pengumpulan sampel harian untuk evaluasi selama 6-7 hari. Namun,
ini membutuhkan waktu dan benar-benar tidak praktis dalam banyak sistem.
Selain itu, keterlambatan dalam pengujian tidak populer, karena menunda awal
musim kawin. Oleh karena itu, dalam praktiknya, sebagian besar evaluasi
dilakukan pada sampel ejakulasi tunggal.

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2.3.1 Evaluasi Tampak Luar
2.3.1.1 Penampilan
Semen kuda jantan biasanya berwarna putih susu dengan
ketebalan setara dengan krim tunggal. Seharusnya tidak
mengandung darah, kontaminasi urin atau gumpalan. Jika ada,
sampel harus dibuang dan kuda jantan diperiksa. Volume semen
normal yang dihasilkan oleh kuda jantan pada setiap ejakulasi
sangat bervariasi 30–250 ml, tetapi, rata-rata sebagian besar
kuda jantan menghasilkan 100 ml dan dari ini, fraksi gel
biasanya 20-40 ml. Namun, variasi yang cukup jelas, baik antara
kuda jantan yang berbeda dan antara koleksi yang berbeda
sepanjang tahun. Kualitas dan kuantitas semen juga bervariasi
dalam musim kawin.
2.3.1.2 pH
Pengukur pH standar dapat digunakan untuk menilai
keasaman/alkalinitas sampel semen. Kondisi asam diketahui
bersifat spermisida. Peningkatan pH juga dapat
mengindikasikan bahan asing atau infeksi. pH 6,9-7,8 dapat
diterima, dengan tingkat 7,3-7,7 menjadi yang terbaik
2.3.2 Evaluasi Mikroskop
2.3.2.1 Extender Semen
Sebelum evaluasi mikroskopis, sampel selalu diperpanjang,
meskipun indikasi motilitas sering dinilai secara mikroskopis,
menggunakan sampel mentah. Pengenceran terutama digunakan
untuk memungkinkan spermatozoon individu diamati, karena
spermatozoa dalam semen mentah cenderung menggumpal
bersama, membuat lebih dari perkiraan pergerakan sangat sulit
sampai mustahil. Perpanjangan sampel juga memperpanjang
umur spermatozoon, memberikan mereka sumber energi
tambahan dan substrat untuk bertahan hidup, dan pemeliharaan
viabilitas saat evaluasi berlangsung. Penambahan ekstender
harus dilakukan segera setelah pengumpulan, idealnya dalam

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 waktu 2 menit, untuk mengurangi kemungkinan mendapatkan
hasil yang salah dari hilangnya viabilitas spermatozoa yang
disebabkan oleh keterlambatan. Ada banyak ekstender yang
tersedia dan digunakan dengan sukses selama proses evaluasi.
Secara umum, ini sama dengan yang digunakan dalam persiapan
semen untuk IB langsung atau penyimpanan dingin. Yang
paling populer adalah yang berdasarkan padatan susu kering
tanpa lemak atau susu skim. Kecepatan pengenceran yang
dibutuhkan tergantung pada konsentrasi sperma dan juga tes
yang dilakukan.

Tabel 1. Contoh ekstender digunakan untuk evaluasi semen.

2.3.2.2 Motilitas
Motilitas sperma dinilai pada skala 0–5, 0 sangat buruk dan
5 sangat baik. Motilitas harus segera dinilai untuk mendapatkan
pengukuran yang akurat. Ini dapat dinilai secara visual atau
melalui sistem analisis motilitas yang terkomputerisasi. Semen
yang tidak dilarutkan dapat diperiksa secara visual di bawah
mikroskop cahaya. Gerakan sampel kemudian dinilai. Atau, itu
dapat dinilai diencerkan, ketika karakteristik motilitas sperma
individu dapat dinilai, meskipun pengenceran itu sendiri dapat
mempengaruhi motilitas. Karenanya penilaian cenderung
subyektif dan tergantung pada pengalaman penguji sebelumnya.
Tabel 2 memberikan panduan kasar untuk nilai (0-5) yang
sering digunakan.

11
 Karena subyektivitas penilaian mikroskopis, metode lain
untuk menilai motilitas telah diselidiki, termasuk metode time-
lapse dark-field photographic, teknik sinematografi dan analisis
komputer. Korelasi antara hasil yang diperoleh dengan analisis
visual dan komputer dilaporkan sangat baik, yaitu 0,92. Analisis
komputer kini telah dikembangkan untuk melakukan evaluasi
penuh, tidak hanya dari motilitas. Biaya peralatan berarti bahwa
analisis tersebut sebagian besar terbatas pada penggunaan
penelitian, meskipun mereka memberikan hasil yang otomatis,
cepat, dan objektif.
Terlepas dari metode penilaian yang digunakan, klasifikasi
jenis gerakan, seperti ditunjukkan dalam Tabel 2, adalah
penting. Diperlukan gerakan progresif; Gerakan osilasi (gerakan
di tempat) atau gerakan dalam lingkaran yang sangat sempit
diklasifikasikan sebagai tidak normal. Jika sperma individu
tidak dinilai, maka pola sperma yang tidak diencerkan hanya
dapat memberikan indikasi pergerakan sperma individu.
Beberapa evaluator mengklasifikasikan motilitas sperma
sebagai rasio mereka yang menunjukkan gerakan progresif :
gerakan osilasi.
Semen yang mengandung setidaknya 40% spermatozoa
motil progresif aktif, yang merupakan grade 2–3, dapat
dianggap memadai untuk IB. Namun, korelasi antara motilitas
dan kesuburan adalah variabel dan rendah (0,7). Meskipun
memberikan korelasi terbaik dengan kesuburan dibandingkan
dengan semua parameter lainnya, itu tidak dapat diandalkan
untuk memberikan indikasi absolut.

12
 Tabel 2. Kriteria untuk menilai pergerakan sperma
2.3.2.3 Longevitas
Penilaian motilitas dari waktu ke waktu dapat digunakan
sebagai indikasi kelayakan. Namun, harus diingat bahwa
longevitas dalam test tube tidak harus sama dengan umur
panjang dalam rahim kuda. Upaya telah dilakukan untuk meniru
longevitas di saluran kuda betina. Penilaian longevitas
melibatkan evaluasi motilitas awal dan kemudian pada berbagai
interval setelah penyimpanan pada 5°C, 22°C atau 37-38°C.
Sebagai contoh, jika motilitas tidak kurang dari 45% setelah 3
jam atau 10% setelah 8 jam pada 22°C, maka semen dapat
diklasifikasikan sebagai cukup baik untuk IB.
2.3.2.4 Konsentrasi
Konsentrasi sperma dalam sampel semen adalah penentu
utama nilainya dan secara tradisional dinilai menggunakan
hemositometer. spectrophotometer manual atau otomatis adalah
perkembangan yang lebih baru dan sekarang dapat membentuk
bagian dari sistem analisis sperma komputer lengkap.
Hemositometer terdiri dari slide penghitung dengan slip
penutup, di mana volume semen yang diencerkan dapat
terperangkap dan dilihat. Jumlah sperma dalam sampel pada
hemositometer dihitung dengan menggunakan grid
penghitungan. Dari angka ini dan tingkat pengenceran,
konsentrasi sperma dapat dihitung. Metode ini memberikan
penilaian yang sangat akurat, tetapi memakan waktu dan tidak
mudah dilakukan di lapangan. Spectrophotometer, di sisi lain,

13
 dapat digunakan di mana saja, tetapi hasil yang diperoleh dapat
bervariasi dan hanya sebaik kalibrasi awal menggunakan
hemositometer. Semen diencerkan sampai sekitar 1 : 30;
pengencer yang digunakan harus jelas secara optik, misalnya,
formalin 10% ditambah salin 0,9%. Jumlah cahaya yang
melewati sampel kemudian dibaca oleh spectrophotometer, dari
mana konsentrasi sperma dihitung.
Kisaran normal untuk konsentrasi sperma adalah 30-600 ×
106 sperma ml − 1 semen yang tidak diencerkan tetapi
konsentrasinya bervariasi secara signifikan. Jumlah total 100 ×
106 sperma yang layak per inseminasi diperlukan untuk tingkat
pembuahan yang dapat diterima. Dalam praktiknya, sampel
dengan konsentrasi 100-200 × 106 ml − 1 dianggap dapat
diterima untuk IB.
2.3.2.5 Morfologi
Pemeriksaan morfologis adalah metode umum untuk
mencoba menilai kesuburan kuda jantan. Sperma dalam sampel
semen encer diperiksa secara mikroskopis. Sperma individual
diperiksa dan persentase sperma abnormal dalam sampel
dicatat. Melihat masing-masing sperma dapat ditingkatkan
menggunakan berbagai pewarnaan, yang memungkinkan
berbagai komponen atau kelainan spermatozoa diidentifikasi,
misalnya, pewarnaan nigrosin-eosin, yang mewarnai kepala
sperma yang mati ungu. Pewarnaan sperma untuk menilai
integritas daerah tertentu juga dapat dilakukan, misalnya
pewarnaan akrosom, uji imuno-fluoresensi dan diberi label
antibodi monoklonal. Prinsip-prinsip metode ini telah
dieksploitasi dalam sistem analisis morfometri spermatozoa
otomatis. Namun, sistem seperti itu adalah spesialis dan mahal
dan dengan demikian tidak sering menjadi bagian dari proses
evaluasi rutin.

14
 Abnormalitas dapat diklasifikasikan sebagai primer
(kegagalan spermatogenesis, kegagalan pematangan sperma),
sekunder (kerusakan sperma yang terjadi pada ejakulasi) dan
tersier (penanganan yang tidak tepat setelah ejakulasi).
Kegagalan spermatogenesis biasanya ditandai oleh sperma
dengan dua kepala, dua ekor, tidak ada bagian tengah, tidak ada
ekor, ekor rudimenter, atau ekor yang sangat melingkar.
Abnormalitas seperti itu mungkin mengindikasikan masalah
jangka panjang atau bahkan permanen. Kegagalan maturasi
ditandai dengan adanya tetesan sitoplasma pada bagian tengah
sperma. Ketika proses pendewasaan berlangsung, tetesan
sitoplasma ini semakin bergerak turun dan menghilang. Jika
mereka hadir dalam sperma ejakulasi, itu merupakan indikasi
bahwa sperma tersebut belum matang. Ini mungkin hanya
sementara, periode istirahat memperbaiki masalah. Kerusakan
yang terjadi pada ejakulasi biasanya ditandai dengan kelainan
ekor, tikungan, gulungan, kekusutan atau pembengkakan,
kepala dan ekor yang terlepas dan tetesan protoplasma.
Akhirnya, kerusakan setelah ejakulasi dimanifestasikan sebagai
hilangnya akrosom, keretakan/ketebalan bagian tengah dan
pecahnya kepala sperma.
Korelasi antara morfologi dan kesuburan relatif rendah
(0,25-0,5). Penelitian telah dilakukan dalam upaya untuk
mengkorelasikan abnormalitas spesifik dengan kesuburan,
tetapi sekali lagi korelasinya buruk. Oleh karena itu, morfologi
bukanlah indikator yang sangat baik tentang potensi kesuburan,
seperti halnya dengan motilitas, jika tidak ada yang lain, maka
digunakan.
Semen yang mengandung 65% atau lebih sperma normal
secara morfologis dianggap sesuai untuk IB.

15
 Gambar 5. Beberapa contoh kelainan yang umum yang mungkin terlihat saat
memeriksa sperma untuk morfologi. Baris atas dari kiri ke kanan: (a)
spermatozoon normal. Cacat akrosom: (b) bengkak, (c) terangkat sebagian, (d)
kecil, (e) terangkat, (f) bagian hilang; cacat kepala: (g) kepala besar, (h)
memanjang, (i) rata, (j) lanciolated, (k) microhead, (l) kepala ganda; cacat leher:
(m) ditekuk, (n) rusak. Baris kedua: cacat bagian tengah: (o) pendek, (p) lemak,
(q) terbelah / menyempit, (r) annulus bengkok, (s) fibula, (t) patah, (u) ganda;
cacat ekor: (v) berbelit-belit, (w) pembuka botol, (x) bengkok, (y) kecil, (z) ganda,
(a1) shoehorn ; tetesan: (b1) proksimal, (c1) distal

2.3.2.6 Ratio Hidup : Mati

Motilitas memberikan indikasi persentase sperma hidup
dalam sampel. Namun, pewarnaan sperma yang berbeda, yang
kemudian diperiksa secara mikroskopis, memberikan hasil yang
lebih akurat. Pewarnaan dengan nigrosin-eosin dalam volume
yang sama dengan semen memungkinkan pewarnaan diferensial
sperma yang mati dan hidup. Sperma yang mati, seperti mereka
permeable ke pewarnaan, muncul sebagai ungu. Pewarnaan lain
telah digunakan dengan sama suksesnya, termasuk etidium
bromida dan fluoresensi akridin oranye atau fluoresensi H25.

16
 Rasio atau persentase sperma mati dan hidup dinilai dalam
sejumlah sampel dari koleksi. Rasio hidup : mati, 6 : 4 (60%
sperma hidup) dapat diterima untuk IB. Namun, sampel dengan
rasio yang lebih rendah dapat dianggap dapat digunakan, jika
memiliki jumlah sperma total yang tinggi, karena dapat
diencerkan lebih sedikit dan ini masih memastikan bahwa
jumlah minimum sperma hidup untuk pembuahan diinseminasi.
2.3.2.7 Sitologi
Sel darah, leukosit dan eritrosit dapat diidentifikasi dalam
sampel semen, menggunakan hematoksilin dan eosin atau
pewarnaan Wright dan dilihat di bawah haemocytometer.
Jumlah leukosit yang lebih besar dari 1500 ml − 1 merupakan
indikasi masalah, mungkin karena infeksi terutama jika pH
semen juga tinggi. Sampel semacam itu tidak akan sesuai untuk
digunakan dalam IB. Konsentrasi eritrosit di atas 500 ml − 1
merupakan indikasi masalah, seperti perdarahan, cedera, dll,
dan juga akan membuat sampel tidak sesuai untuk digunakan
dalam IB.
2.3.2.8 Bakteriologi
Sampel semen berpotensi mengandung bakteri, baik patogen
maupun non-patogen. Isolasi dan identifikasi bakteri patogen
khususnya diperlukan untuk mencegah lewatnya infeksi saat
inseminasi. Bakteri dapat diidentifikasi dengan kultur langsung
sampel semen ke piring agar atau dengan penyeka swab yang
diambil dari semen atau genitalia. Inkubasi dalam berbagai
kondisi memungkinkan diferensiasi bakteri. Infeksi jangka
panjang atau akut juga dapat terbukti karena jumlah leukosit
yang tinggi dalam sampel semen atau bukti nanah. Identifikasi
bakteri seperti Pseudomonas aeruginosa, Taylorella
equigenitalis dan Klebsiella pneumoniae kemungkinan akan
menghalangi sampel semen untuk digunakan.

17
2.4. Penyimpanan dan Penggunaan Semen pada Kuda
Setelah semen dievaluasi, semen dapat dipertimbangkan untuk inseminasi.
Semen dapat digunakan dalam satu dari empat cara:
1. Digunakan segera tanpa dilarutkan untuk menginseminasi satu atau
mungkin dua kuda betina, tergantung pada volume yang dikumpulkan.
2. Diencerkan dan segera digunakan untuk inseminasi beberapa kuda betina.
3. Diencerkan dan didinginkan untuk digunakan selama 72 jam berikutnya.
4. Diencerkan dan dibekukan untuk digunakan di kemudian hari.
Metode yang digunakan tergantung pada sistem stud dan lokasi kuda
betina yang akan diinseminasi.
2.4.1 Semen Mentah
Hasil terbaik diperoleh dengan menggunakan semen murni yang
diinseminasi segera. Namun, penggunaan semen seperti itu menyalahkan
dua tujuan utama IB yaitu meningkatkan jumlah kuda Betina yang dapat
dibuahi dengan satu ejakulasi dan transportasi, meskipun mungkin dapat
digunakan sebagai bantuan dokter hewan atau manajemen.
2.4.2 Semen Dingin
Jika semen akan diperpanjang sebelum digunakan, itu harus segera
dilakukan. Sejumlah besar pengencer telah dikembangkan, biasanya
pada susu, gelatin atau kuning telur ditambah antibiotik.
Semen yang diperpanjang dapat disimpan hingga 2-3 hari jika
diperpanjang dalam perbandingan 2 : 1, semen : ekstender dan
didinginkan perlahan hingga 4-8°C selama 4 jam dan disimpan pada suhu
ini sampai digunakan. Perawatan semacam itu memungkinkan
transportasi dan penyimpanan semen terbatas dalam lemari es atau
Equitainer, selama suhunya dijaga pada suhu 4°C. Equitainer diatur
sedemikian rupa untuk memastikan penurunan bertahap suhu semen
−0,3°C min − 1 hingga 4–5°C. Ini kemudian akan menjaga semen pada
suhu ini hingga 24-48 jam.
2.4.3 Semen Beku
Penyimpanan berkepanjangan hanya dapat dicapai dengan
pembekuan, tetapi teknik pembekuan semen kuda tidak pernah seperti

18
 pada ternak. Masalah utama adalah mengidentifikasi cryoprotectant yang
cocok yang dapat digunakan untuk memperpanjang pembentukan kristal
es baik di dalam kepala sperma, sehingga mengurangi kerusakan fisik,
atau mengurangi pengeringan sperma. Gliserol digunakan sebagai agen
pada ternak, tetapi tampaknya beracun untuk sperma kuda. Namun,
dengan tidak adanya agen sukses lainnya, konsentrasi rendah gliserol
terus digunakan. Deterjen dan kombinasi gula juga telah digunakan
sebagai cryoprotectants. Selain variasi hasil dengan ekstender, ada
variasi besar antara dan dalam kuda jantan, dengan tingkat kehamilan 10-
70% dilaporkan.

2.5. Pengenceran Semen pada Kuda

Tingkat pengenceran semen tergantung pada konsentrasi awal sampel dan
motilitas sperma. Inseminasi semen encer yang mengandung 100 × 106 sperma
motil progresif (PMS) per inseminasi memberikan hasil yang baik, tetapi
biasanya 500 × 106 sperma per inseminasi direkomendasikan untuk
memungkinkan margin of error. Volume inseminasi dihitung menggunakan
rumus berikut:

Inseminasi 800 × 106 sperma disarankan saat menggunakan semen beku,

untuk mengkompensasi kehilangan yang terjadi selama proses pembekuan.

2.6. Volume Inseminasi Buatan pada Kuda

Volume inseminasi bervariasi dari 10 hingga 30 ml untuk semen segar, 10
hingga 60 ml untuk dingin dan 0,5 hingga 5 ml untuk beku. Telah disarankan
bahwa volume lebih dari 100 ml atau kurang dari 0,5 ml merusak tingkat
pembuahan.

19
2.7. Teknik Inseminasi Buatan pada Kuda
Kuda betina diinseminasi tanpa operasi. Ketika menggunakan semen segar
atau dingin, ini harus terjadi, seperti dengan layanan alami, pada hari kedua
atau hari ke-4 dari estrus. Semen beku membutuhkan sinkronisasi yang lebih
baik dengan ovulasi, idealnya dalam waktu 6 jam. Semen baik diencerkan dan
tidak diencerkan disimpan ke dalam rahim melalui pipet steril plastik dengan
jarum suntik terpasang atau dengan gun inseminasi dipandu masuk melalui
serviks ke rahim menggunakan jari telunjuk. Atau, pipet dapat diarahkan
melalui serviks sesuai palpasi rektum, serviks dirasakan melalui dinding
rektum.
Jarum suntik diisi dengan semen yang dipegang di antara dua gelembung
udara dan kemudian ditempelkan di ujung pipet. Begitu melewati serviks, pipet
inseminasi didorong ke dalam rahim sekitar 2 cm. Ketika sudah di tempat,
semen perlahan-lahan dikeluarkan dengan menekan plunger.

20
 BAB III PENUTUP

3.1. Simpulan
Di berbagai belahan dunia, IB kuda tersebar luas dalam penggunaannya.
Inggris, meskipun merupakan pemimpin dalam banyak aspek industri kuda,
tertinggal, sebagian besar karena kegagalan industri Throughbred untuk
mengenali dan karenanya mendaftarkan keturunan yang dikandung oleh IB.
Sampai dapat dibujuk bahwa IB adalah cara yang dapat diterima untuk
membiakkan kuda, penerapan IB akan dibatasi untuk digunakan dalam
masyarakat peternak lain. Meskipun demikian, jelas bahwa disini kuda ada di
sini untuk tinggal dan akan terus berkembang, membuka dengan itu peluang
menarik dalam pemilihan dan pemuliaan spesies kuda.

3.2. Saran
Saran dari penulis untuk penulisan ini masih jauh dari kata sempurna,
masih banyak sumber-sumber yang lebih compatible mengenai materi ini.
dimohonkan para pembaca tidak hanya menggunakan paper ini sebagai acuan
tetapi bisa mencari di tempat lain dengan informasi yang lebih lengkap.

21
 DAFTAR PUSTAKA

M.C.G. Davies Morel, 2003, Equine Reproductive Physiology, Breeding and Stud
Management 2nd Edition, CABI Publishing, Institute of Rural Studies
University of Wales, Aberystwyt, UK

Guilherme Pugliesi, Giovanni Ribeiro de Carvalho, Daniel Macêdo Rates, Pedro

Gama Ker, Manuela Pereira da Matta, Renan Reis de Oliveira, José
Monteiro da Silva Filho, 2012, Viability and fertility of cooled equine semen
diluted with skimmed milk or glycine egg yolk-based extenders, R. Bras.
Zootec., vol. 41, no. 12, hh. 2411-2417.

H. Siemea, A. Bonk, H. Hamann, E. Klug, T. Katila 2004, Effects of different

artificial insemination techniques and sperm doses on fertility of normal
mares and mares with abnormal reproductive history, Theriogenology, vol.
62, hh. 915–928.

Alicja Kowalczyk, Ewa Czerniawska-Pia˛tkowska and Marian Kuczaj 2019,

Factors Influencing the Popularity of Artificial Insemination of Mares in
Europe, Animals, vol. 9.

22
 Revista Brasileira de Zootecnia
© 2012 Sociedade Brasileira de Zootecnia
ISSN 1806-9290 R. Bras. Zootec., v.41, n.12, p.2411-2417, 2012
www.sbz.org.br

Viability and fertility of cooled equine semen diluted with skimmed milk or
glycine egg yolk-based extenders

Guilherme Pugliesi1, Giovanni Ribeiro de Carvalho1, Daniel Macêdo Rates2, Pedro Gama Ker1,
Manuela Pereira da Matta2, Renan Reis de Oliveira1, José Monteiro da Silva Filho3

1
Department of Animal Science, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
2
Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
3
Department of Veterinary Clinics and Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

ABSTRACT - Two semen extenders were compared for their ability to maintain viability of horse semen during 24 hours
of cold preservation, and for the pregnancy rate after artificial insemination. In the experiment 1, five ejaculates from three
stallions were split-diluted in either a skimmed milk-based extender (Kenney extender) or a glycine egg yolk-based extender
(Foote extender) and cooled at 6–8 °C for 24 hours. Semen samples stored in Kenney extender for 24 hours had higher motility
and spermatic vigor compared with those stored in Foote extender. However, samples stored in Foote extender had higher
number of reactive sperm by hypoosmotic test and greater viability by epifluorescence test compared with those in Kenney
extender. In the experiment 2, 17 and 23 ejaculates from two stallions were split-diluted with Kenney extender and Foote
extender. The sperm concentration in each extender was adjusted to 500 million viable sperms per insemination dose. Semen
was cooled to 6–8 °C and stored for 24 hours. Seventy-four cycles of crossbred mares were inseminated with either semen
diluted in Kenney extender or semen diluted in Foote extender. The pregnancy rate was higher from semen diluted in Kenney
extender than that from semen in Foote extender (0.553 vs. 0.306). The Kenney extender is effective in preserving the motility,
vigor and fertility of stallion semen after 24 hours of cold storage, whereas the Foote extender is not acceptable.

Key Words: artificial insemination, cooling, semen extender, stallion

Introduction and boar semen to cold shock during storage at low

Artificial insemination with cooled semen has been
routinely used in equine reproduction as it allows higher
fertility rates compared with those with frozen semen,
when used within 24 hours after collection (Jasko et al.,
1992; Vidament et al., 1997; Aurich & Aurich, 2006).
The semen extenders used for dilution and packaging
of equine cooled semen are mostly based on skimmed milk
and egg yolk. The active components involved in sperm
protection by milk are casein micelles, which interact
with membrane plasma proteins and protect against the
removal of phospholipids and cholesterol from the plasma
membrane (Bergeron & Manjunath, 2006). The egg yolk, in
combination with other components of extender, can help
the sperms to resist the cold shock (Bogart & Mayer, 1950;
Amirat et al., 2004) and this protective action is mainly
attributed to low-density lipoprotein (Pace & Graham,
1974; Moussa et al., 2002).
The sperm plasma membrane in stallion and boar
has a lower ratio of cholesterol to phospholipids than the
bull sperm plasma membrane (Parks & Lynch, 1992).
This apparently results in more susceptibility of stallion
temperatures (Parks & Lynch, 1992). In this regard, the
semen extenders that are commonly used for cooling of
boar semen may be adapted for use in storage of stallion
cooled semen for better fertility results. A glycine egg
yolk-based extender (Foote, 2002) was used for cooling
of boar semen and the sperm motility did not decline more
than 10% during 2 days of storage at various temperatures
(5, 15 or 25 °C). The same extender maintained the fertility
of donkey semen after dilution and cooling at 5 °C for 12
hours (Rossi et al., 2012), showing promising results for
adaptation for use in cooling of stallion semen.
An estimate of potential fertility of semen sample can
be obtained through several in vitro tests for evaluation
of semen characteristics (Mocé & Graham, 2008). Semen
evaluation using these laboratory tests is rapid, inexpensive
and extremely important for the artificial insemination
practice to ensure high semen quality.
The objective of the present study was to evaluate
and compare the in vitro viability and in vivo fertility of
stallion semen stored at low temperature (6–8 °C) for 24
hours using an extender (Kenney extender, a skimmed
powdered milk-based extender; Kenney et al., 1975) that is

Received October 5, 2011 and accepted August 3, 2012.

Corresponding author: pugliesi_vet@hotmail.com
2412 Pugliesi et al.
commonly used in stallion semen dilution and an extender
(Foote extender, a glycine-egg yolk-based extender; Foote,
2002) that is used for boar semen.
Material and Methods
In experiment 1, three stallions of the Mangalarga
Marchador breed, 8 to 14 years-old, were selected with
no apparent abnormalities of the reproductive tract, as
determined on the basis of andrological examination and
reproductive history, and kept in 5 × 3 m stalls. Stallions were
fed elephant grass (Pennisetum purpureum), concentrate
(corn and soybean), and mineral salt and were in good
body condition during the period of semen collection for
the experiment.
Semen samples were collected in December during the
reproductive season in Brazil. Five ejaculates per stallions
were used, collected three times a week with the aid of an
artificial vagina (adapted Hannover model) using a mare in
estrus as dummy. The ejaculates were immediately taken
to the laboratory for further processing. Each ejaculate was
split into two parts. One part was diluted in Kenney extender,
a skimmed milk based extender (Kenney et al., 1975),
commonly used for dilution of stallion semen. The other
part was diluted in Foote extender, a glycine-egg yolk based
extender (Foote, 2002) used for dilution of boar semen.
After the dilution, both the parts were packaged in aliquots
containing 12 mL of diluted semen with final concentration
of 30 × 106 viable sperm/mL, and stored in a container
(Equitainer®; Hamilton Research, Inc. 66 Woodland Mead,
South Hamilton, MA, USA) at 6-8 °C for 24 hours.
The semen quality was evaluated for fresh, diluted and
cooled semen. The physical parameters of motility (number
of sperm with progressive movement/100 sperm cells) and
vigor (status of the sperm motility), scored on a scale from
0 (without movement) to 5 (fast progressive movement),
were evaluated under a light microscope (400×) by two
trained evaluators who did not know the treatment groups.
The sperm morphology of diluted and cooled semen was
evaluated after deposition of one drop of semen, previously
diluted in saline formaldehyde solution buffered at 37 °C,
between slide and cover slip. Counting of 200 sperm cells
was done under an optical phase contrast microscope
(1000×) for enumeration of different types of morphological
abnormalities (Brito, 2007; Varner, 2008) and subsequent
classification for minor or major defects (Blom, 1972).
The integrity of the plasma membrane was evaluated by
supravital test and functionality by hyposmotic test (HOST)
in the diluted semen before and after the cold storage. The
supravital test was done using eosin-nigrosin stain. One
drop (10 µL) of semen was placed together with one drop
(10 µL) of dye on a slide, and a smear was made after 2
minutes on a hot plate at 37 °C. The sperms classified as
intact (viable) membrane were not stained by eosin, while
the non-intact (nonviable) membrane showed the pink-red
stained nuclei. From each one, a sample of 100 sperm cells
were counted under common optical microscope (400×).
For HOST, a solution of 100 mOsmol sucrose-base
was used. Aliquots of 1 mL of this solution were placed
in tubes at 37 °C and 20 µL of semen sample were added
and incubated for 50 minutes in a water bath at 37 °C.
Subsequently, the sperms were analyzed by counting 100
sperm cells from each sample in phase contrast microscopy
(400×). The cells were classified by the presence or absence
of coiled tail (Neild, 1999). The calculations of hyposmotic
reaction were done using the formula (reactive sperms by
HOST = % of coiled tails after HOST – % of coiled tails
before HOST) described by Melo & Henry (1999).
The assessment of membrane integrity of the semen
after 24 hours of cooling was also verified using the
combination of two fluorescent dyes: propidium iodide
(PI) and carboxyfluorescein diacetate (CFDA). Sperm
showing complete or partial red fluorescence (PI staining)
was considered nonviable, while sperm showing green
fluorescence was considered viable (Brito, 2007). For
CFDA/PI staining, 10 µL of diluted semen after cold storage
for 24 hours at 6–8 °C was added to a conical tube containing
40 mL of stock solution of CFDA/PI in a dark room at 25 °C.
The reading was performed after the homogenization of the
conical tube. The slides were prepared (wet preparation) at
room temperature and evaluated in fluorescence microscope
(1250×), using filters of 480 to 610 nm (fluorescein and
rhodamine, respectively).
In experiment 2, the experiment was done from October
to February during the reproductive season in Brazil. Semen
ejaculates from 2 Mangalarga Marchador breed stallions, at
9 (Stallion 1) and 8 (Stallion 2) years of age, were used.
The selection, feeding and management were similar to
experiment 1.
To determine the fertility rate of the diluted and cold-
stored ejaculates after artificial insemination, 38 crossbred
Breton-Postier mares, of 4 to 18 years of age, weighing 350
to 500 kg and with good body condition score were used.
The mares were selected with no apparent abnormalities
of the reproductive tract, as determined by ultrasound
examinations and reproductive history (Ginther, 1995). The
animals were kept in paddocks. Mares received mineral
salt ad libitum and supplementation with shopped elephant
grass (Pennisetum purpureum) and concentrate (corn and
soybean).
R. Bras. Zootec., v.41, n.12, p.2411-2417, 2012
Viability and fertility of cooled equine semen diluted with skimmed milk or glycine egg yolk-based extenders
Seventeen ejaculates from Stallion 1 and 23 ejaculates
from Stallion 2 were collected. The semen collections were
done three times a week (Monday, Wednesday, and Friday),
with the aid of an artificial vagina (adapted Hannover
model) using a mare in estrus as dummy. After collection,
the ejaculates were immediately taken to the laboratory for
further processing. Each ejaculate was split into two parts
and diluted either in Kenney or Foote extender. The dilution
with extenders was performed as described in Experiment 1.
The concentration of sperm was adjusted to 500 million
viable sperms per insemination dose in a final volume of
15 mL. The semen was placed in Equitainer® for cooling at
6-8 °C for 24 hours. The mares were inseminated by semen
dose either with Kenney or with Foote extender. Semen
from two stallions was used randomly.
The semen quality was evaluated for fresh, diluted and
cooled semen for physical and morphological characteristics
as described in experiment 1.
The mares were regularly observed and examined, and
those which showed estrous signs and/or a follicle greater
than 25 mm in diameter, with good uterine conditions (no
fluid in the uterine lumen and onset of endometrial edema)
were daily monitored by transrectal palpation. After the
detection of 30 mm follicles, mares were randomly assigned
to two groups: mares inseminated with cooled semen in
Kenney extender and mares inseminated with cooled semen
in Foote extender. The semen from both stallions was
randomly used among the mares. Inseminations were made
only on predetermined days (3 times a week - Tuesdays,
Thursdays and Saturdays). The semen was deposited in
uterine body on the day of detection of a 30-40 mm follicle
and repeated until observation of ovulation. Seventy-four
estrous cycles from 38 mares were used. The pregnancy
diagnosis was made from the 10th to the 13th day after the
ovulation by transrectal ultrasonography (Ginther, 1995).
The dependent variables considered were: progressive
motility, sperm vigor, sperm morphology, reactivity
to HOST, number of viable and nonviable sperms by
Table 1 - Mean of data for physical and morphological characteristics of stallion semen diluted in Kenney extender or Foote extender and
stored at 6–8 °C for 24 hours
Parameter (n=15)
Progressive motility (sperm/100 sperm)
Sperm vigor (0–5)
Normal sperm (sperm/100 sperm)
Major defects (sperm/100 sperm)
Minor defects (sperm/100 sperm)
a,b - Means with different letters (a, b) in the same row within each semen category (diluted or cooled) differ (P<0.05).
x,y - Means with different letters (x, y) in the same row within each semen extender (Kenney or Foote) differ (P<0.05).
1
Semen before cooling.
2
Semen after cooling at 6–8 °C for 24 hours.
2413
supravital dye test and number of viable and nonviable
sperms by epifluorescence dye test. The data that showed
a normal distribution by Shapiro-Wilk test were compared
using ANOVA followed by the Student-Newman Keuls test
for comparisons between extenders and between stallions.
Data that were not normally distributed (spermatic vigor)
were transformed to log. The simple Pearson correlation
was tested among the variables (progressive motility,
reactive to HOST, viable by epifluorescence and viable by
supravital test) to verify the possible relationships between
the characteristics after cooling. Pregnancy rate in mares
was compared by the frequency distribution of Chi-square
(X²) to check for possible differences between extenders.
The statistical software SAS was used (SAS Institute Inc,
version 9.2). The data are given as the mean±SD unless
otherwise indicated. A probability of P<0.05 indicated that
the difference was significant.
Results and Discussion
In experiment 1, the mean values of progressive
motility and sperm vigor of the fresh semen were 62.7±9.3
sperm/100 sperm and 3.2±0.3, respectively and did not
differ between stallions. For diluted semen before cold
storage, there were no differences in any physical and
morphological characteristics of sperms between the
Kenney and Foote extenders (Table 1). For cooled semen
(6-8 °C for 24 hours), progressive motility and sperm
vigor of semen diluted with Kenney extender were higher
(P<0.05) than that of semen diluted with Foote extender;
no difference was found between the extenders in any
sperm morphology characteristics. The percentage of
sperm defects was higher (P<0.05) in the diluted semen
after than before cooling, irrespective of extender.
In experiment 2, the mean values of progressive
motility and sperm vigor of the fresh semen were 69.9±5.0%
and 3.4±0.3, respectively, and did not differ between
stallions. The mean percentage of progressive motility
Diluted1 Cooled2
Kenney Foote Kenney Foote
62.3±9.6 60.0±11.0 37.0±9.4a 30.3±11.0b
3.4±0.4 3.1±0.3 2.4±0.3a 2.0±0.5b
71.3±7.5x 72.0±8.8x 65.7±8.7y 67.7±9.8y
8.8±3.8 7.1±3.0 9.3±5.3 7.9±5.0
20.0±7.8y 22.2±9.7y 25.0±7.8x 24.6±10.9x
R. Bras. Zootec., v.41, n.12, p.2411-2417, 2012
2414
and sperm vigor of cooled semen were higher (P<0.05) in
Kenney extender (51.0±10.5 sperm/100 sperm and 3.0±0.4,
respectively) than that in Foote extender (42.5±9.9 sperm/
100 sperm and 2.4±0.5, respectively). The mean values of
number of normal sperm/100 sperm in fresh semen (before
dilution), semen cooled with Kenney extender, and semen
cooled with Foote extender were 73.4±4.3; 63.0±11.1;
and 64.2±14.5, respectively. The mean number of normal
sperm/100 sperm after cooling at 6–8 °C for 24 hours was
lower (P<0.05) in both extenders by the morphology test,
but there was no significant difference between extenders.
After cooling for 24 hours, the semen diluted in both
extenders showed a higher (P<0.05) number of sperm
with minor defects compared with those of fresh semen
(unpublished data).
The significant higher progressive motility and sperm
vigor obtained with Kenney extender in both experiments
is probably due to better availability of substrate in
this extender (glucose-skimmed milk) in comparison
with Foote extender (glycine-egg yolk), a characteristic
that provides greater energy support for survival and
movement of spermatic cell. Changes in organization of the
plasma membrane fluid mosaic can lead to alterations in
permeability, functionality and metabolism of sperm cell,
which may affect the motility and fertilizing ability (Amann
& Graham, 1993). Although the protection mechanism
of milk for the sperm is not clearly known, it is believed
that phosphocaseinate and β-lactoglobulin are the major
components of milk that promote this protection (Batellier
et al., 1998; 2001). Another factor that may be correlated to
the survival of sperm cells during storage is the amount of
energy reserves contained in extender and seminal plasma,
which, in the case of exhaustion of reserves, could promote
sperm metabolic death, since motility is the process that
requires more energy and has a positive correlation with
ATP concentrations (Heisnaken et al., 1987; Januaskauskas
& Rodriguez-Martinez, 1995). The previous studies using
egg yolk in equine semen extenders were equivocal. Some
studies (Province et al., 1984; Bruemmer et al., 2002)
Table 2 - Mean of data for the number of reactive sperm by HOST, and viable sperm by supravital and epifluorescence test in stallion semen
diluted in Kenney extender or Foote extender and stored at 6–8 °C for 24 hours
Parameter (n=15)3
HOST (reactive sperm/100 sperm)
Supravital (viable sperm/100 sperm)
Epifluorescence (viable sperm/100 sperm)
Means with different letters in the same row within each semen category (diluted or cooled) differ (P<0.05).
HOST - hyposmotic test.
1
Semen before cooling.
2
Semen after cooling at 6–8 °C for 24 hours.
3
For epifluorescence test for cooled semen n=12.
Pugliesi et al.
showed inferiority of egg yolk-based extenders and others
(Rota et al., 2004; Melo et al., 2005; Rota et al., 2008)
showed superiority of yolk-based extenders on the motility
and viability at different periods of storage in comparison
with those from milk-based extenders.
Although there was no difference in the number
of normal and defective sperms between extenders
after cooling in both experiments, the number of sperm
defects increased with both extenders compared with
fresh diluted semen, and this reinforces the previous
studies (Graham, 1996) in which the cooling process
promoted irreversible cellular injuries, which may impair
the semen fertility. However, the increase was mostly
in minor defects and could have been promoted by cold
shock. These defects are considered less important than
the major defects when considering fertility, and can be
compensated by increasing sperm number in insemination
dose (Chenoweth, 2005).
In experiment 1, there were no significant differences
in the functionality of sperm plasma membrane by HOST
and integrity by supravital staining in diluted semen before
cooling between Kenney and Foote extenders (Table 2).
The data for epifluorescence test on cooled semen was
not available in 3 samples in each extender due to lack
of semen evaluation within two hours after staining with
the fluorescent probes. For cooled semen after 24 hours
at 6-8 °C, sperm functionality by HOST and the integrity
by epifluoresence was higher (P<0.05) in Foote extender
compared with that of Kenney extender; the supravital
viability did not differ between extenders. A significant
(P<0.05) correlation was observed between HOST and
epifluorescence test (r = 0.46) and between HOST and
supravital test (r = 0.39) combined for extenders (data not
shown). No correlations were observed between any other
characteristics.
The plasma membrane allows selective transport of
important molecules to the sperm cytoplasm and its integrity
is important for the reactions necessary for union of male
and female gametes (Jeyendran et al., 1984). The two
Diluted1 Cooled2
Kenney Foote Kenney Foote
47.5±15.7 59.2±13.3 23.6±11.3a 39.9±14.0b
61.6±5.3 72.8±8.6 34.9±22.9 47.8±21.4
— — 22.5±20.3a 61.0±14.0b
R. Bras. Zootec., v.41, n.12, p.2411-2417, 2012
Viability and fertility of cooled equine semen diluted with skimmed milk or glycine egg yolk-based extenders
extenders were similar in their effect on plasma membrane
integrity of cooled sperm by supravital test. However, other
studies have shown superiority in the number of viable
sperm (range 72 to 90 sperm/100 sperm) by supravital test
in extenders containing egg yolk (Melo & Henry, 1999;
Nunes et al., 2008). The superiority of Foote extender by
the HOST and epifluorescence test suggested that it may
give better protection to the plasma membrane during
cooling. However, this apparently could not compensate
the simultaneous impairment of the motility and vigor of
the sperms as shown by the lower pregnancy rate from the
semen diluted in Foote extender (Table 3). The decrease
in motility in the cooled semen in Foote extender may be
attributed to changes in available energy or damage to
elements of axoneme that may compromise progressive
sperm motility without promoting high injury rate in the
plasma membrane (Watson, 1995).
The sperm motility and morphology were the
characteristics that best correlated to fertility of the semen
in some studies (Dowsett et al., 1984; Daels et al., 1991; Jasko
et al., 1992). In addition, in another study (Johansson et al.,
2008) a positive correlation between sperm viability by
supravital test and sperm motility were observed in stallion
semen. In contrast with the present study, an association
between sperm motility and integrity or functionality of
its plasma membrane was not found. In this regard, for an
effective evaluation of semen samples, several in vitro tests
for different sperm characteristics are required. Since the
process of fertilization of an oocyte by a sperm is complex,
an in vitro test for measuring a single attribute of sperm
may not be a foolproof method to assess the quality of
semen. Further studies are indicated for the comprehensive
examination of different forms of damage in the sperm
during the cooling that impair the fertility of the semen.
The pregnancy rate did not differ significantly between
stallions 1 and 2, combined for the two extenders (Table 3).
Thus, data from the two stallions was combined. The
pregnancy rate was higher (P<0.05) for semen with Kenney
extender than that for Foote extender.
Table 3 - Pregnancy rates (number of pregnancy/cycle) of mares
inseminated with stallion semen diluted and cooled in
Kenney extender or Foote extender at 6–8 °C for 24
hours
Kenney (n=38) Foote (n=36)
Stallion 1 0.438 (7/16) 0.267 (4/15)
Stallion 2 0.636 (14/22) 0.333 (7/21)
Overall 0.553a (21/38) 0.306b (11/36)
Percentages with different letters in the same row differ (P<0.05).
2415
The higher pregnancy rate observed with the Kenney
extender (21/38 cycles) compared with the Foote extender
(11/36 cycles) is consistent with higher motility and
vigor observed in vitro for the Kenney extender in both
experiments and indicates a better relationship between
these characteristics with fertility than plasma membrane
assessments. The lower pregnancy rate in semen diluted
with Foote extender could also be due to presence of
egg yolk, which has been shown to contain progesterone
(mean of immunoreactive progesterone, 13.7±2.9 mg/yolk;
Möstl et al., 2001). Progesterone could lead to a premature
sperm capacitation, leading to reduced fertility. Progesterone
can bind to receptors on plasma membrane of equine sperm
(Cheng et al., 1998), an important step in the acrosomal
reaction process. The fertility obtained in this study with
Kenney extender was similar to those obtained in many
studies using milk-based extenders (Douglas-Hamilton et al.,
1984; Jasko et al., 1992; Batellier et al., 1998).
The pregnancy rate for the semen with Foote extender
was lower compared with that reported by others (Carvalho
et al., 1997; Silva Filho et al., 1998; Melo et al., 2005) who
also used egg yolk-based extenders. Thus, adaptations or
modifications of Foote extender for the dilution of stallion
semen may be a topic for further studies so as to improve
the maintenance and protective ability of this semen
extender. The possible modifications could be the addition
of larger amounts of an energy constituent such as glucose
to provide more energy in the form of ATP and thus avoid
the metabolic death of the sperm.
Conclusions
Foote extender, a glycine-egg yolk-based semen
extender, is not effective in preserving the semen
characteristics (progressive motility and sperm vigor) and
does not give acceptable fertility rate after insemination
with diluted equine semen cooled to 6-8 °C for 24 hours.
On the other hand, the Kenney extender is a superior
option for diluting equine semen in programs of artificial
insemination with cooled semen.
Acknowledgments
This experiment was carried out at the Equine Sector of
Universidade Federal de Viçosa, Brazil, with support from
CNPq, FAPEMIG and CAPES. The authors thank Dr. M. A.
Beg, from University of Wisconsin for suggestions and Dr.
A. M. Borges, from Universidade Federal de Minas Gerais
for assistance with measurement of extender osmolarity.
R. Bras. Zootec., v.41, n.12, p.2411-2417, 2012
2416 Pugliesi et al.
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Effects of artificial insemination techniques and sperm doses

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Article  in  Theriogenology · September 2004

DOI: 10.1016/j.theriogenology.2003.12.011 · Source: PubMed

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 Theriogenology 62 (2004) 915–928

Effects of different artificial insemination techniques

and sperm doses on fertility of normal mares and
mares with abnormal reproductive history
H. Siemea,b,*, A. Bonkc, H. Hamannd, E. Klugc, T. Katilae
a
National Stud of Lower Saxony, Spoerckenstr. 10, 29227 Celle, Germany
b
Institute for Reproductive Medicine, Veterinary School Hanover, Hanover, Germany
c
Clinic for Horses, Veterinary School Hanover, Hanover, Germany
d
Institute for Animal Breeding Science, Veterinary School Hanover, Hanover, Germany
e
Department of Clinical Veterinary Sciences, University of Helsinki, Helsinki, Finland
Received 24 July 2003; received in revised form 21 October 2003; accepted 14 December 2003

Abstract

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates
of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using
fresh or frozen–thawed sperm were investigated. The material included 187 normal mares (148
foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history.
Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine
body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or
hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method
of semen (fresh, frozen–thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50
106 or 300
106
progressively motile sperm (pms) for fresh semen and 100 or 800
106 frozen–thawed sperm with
>35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG adminis-
tration when fresh semen was used, or 30 h for frozen–thawed semen. Differences in pregnancy rates
between treatment groups were analyzed by Chi-squared test, and for most relevant factors
(insemination technique, mare, semen, and stallion) expectation values and confidence intervals
were calculated using multivariate logistic models.
Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects
(P > 0:05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between
insemination technique, semen parameters, and mares did have significant effects (P < 0:05). In
problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI
(16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares,
hysteroscopic AI with fresh semen gave significantly (P < 0:05) better pregnancy rates than uterine
body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly

*
Corresponding author. Tel.: þ49-5141-929433; fax: þ49-5141-909746.
E-mail address: stallions.celle@t-online.de (H. Sieme).

0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2003.12.011
916 H. Sieme et al. / Theriogenology 62 (2004) 915–928

lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results
demonstrate that for problem mares, conventional insemination into the uterine body appears to
be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be
expected by hysteroscopic insemination.
# 2004 Elsevier Inc. All rights reserved.

Keywords: Low-dose insemination; Frozen semen; Fresh semen; Hysteroscopic insemination; Pregnancy rate

1. Introduction

A mare’s reproductive history provides valuable information in stud farm practice.

Commonly used terms for the reproductive status of mares are maiden (often divided into
young and old maiden), barren, and foaling [1–3]. Mare age and status have been shown to
affect fertility. Old mares have lowered pregnancy rates per cycle and increased pregnancy
loss rates [3,4]. Moreover, management factors and qualities of the stallion (e.g. sperm
quality, sperm dose, and storage method) affect the reproductive efficiency of mares. The
use of frozen stallion semen in particular warrants careful consideration of the mare’s
reproductive status, as frozen semen in barren [5,6] or old maiden mares [7] may result in
disappointing pregnancy rates.
Mares are typically inseminated with 500
106 fresh progressively motile sperm (pms)
every other day during estrus [8]. For maximum reproductive efficiency, Pickett et al. [9]
recommend for each insemination dose 500
106 fresh pms at the stud farm,
1000
106 pms for shipped semen at the time when the semen is packed before cooling,
and 800
106 total frozen sperm with at least 30% post-thaw progressive motility. A dose
of 50
106 pms has been regarded as the critical number of sperm for fresh semen [10].
Although the minimum number of sperm per insemination dose probably varies between
stallions, it would be useful to determine the lowest sperm numbers needed to produce
commercially acceptable pregnancy rates in the majority of stallions and to evaluate
whether any interaction exists between sperm dose and reproductive status of mares.
Low-dose insemination techniques were initially developed for the use of sex-sorted
sperm because the speed of sorting is insufficient to produce conventional AI doses for
routine uterine body inseminations [11]. Deep uterine horn insemination in the horn
ipsilateral to the preovulatory follicle is the current method of choice for low-dose AI.
Hysteroscopic insemination—depositing semen onto the papilla of the uterotubal junction
(UTJ) using a video-endoscope—seems to yield good results with low sperm numbers [12–
17]. Another promising method is transrectally guided deep uterine horn AI. The position
of a flexible but rigid catheter at the tip of the uterine horn is verified by ultrasound [18] or
by transrectal palpation [19,20]. Deep uterine horn AI has been reported to give similar or
better pregnancy rates than routine AI into the uterine body [19,21–23], but the opposite
claim has also been made [12,18,24]. Because of the contradictory findings, recommend-
ing a single AI method for practice is problematic. In the above-mentioned studies, such
factors as type of semen used and sperm dose, hysteroscopic insemination technique, or
features of the mares may have been different and their effects should be evaluated
separately.
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 917

Low-dose insemination techniques could improve efficiency of frozen stallion semen

since the number of AI doses per ejaculate would increase markedly. The pregnancy rates
of mares after frozen semen AI are relatively poor compared with fresh or chilled semen AI
[9] and are believed to result from damage suffered by sperm during the freezing and
thawing processes [25]. Furthermore, seminal plasma, which is removed before semen
freezing has been suggested to protect sperm in the genital tract of the mare [26].
Therefore, placement of sperm close to the UTJ might increase the number of viable
sperm within the oviduct when frozen–thawed semen is used. Indeed, pregnancies have
been produced using as little as 5
106 frozen pms [15,27].
Insemination of mares is followed by post-breeding endometritis and most of this
reaction is due to the sperm component rather than seminal plasma or extender [28,29]. The
use of low numbers and volumes of sperm directly on the uterotubal papilla has been
suggested to reduce post-breeding endometritis [30], which is particularly important when
inseminating mares with an abnormal reproductive history.
The hypothesis here was that either sperm dose or site/technique of insemination
influences pregnancy rates in normal mares differently than in mares with an abnormal
reproductive history. Our objective was thus to investigate the effects on pregnancy of the
following techniques: routine insemination into the uterine body, rectally guided deep
intracornual insemination, and hysteroscopic insemination onto the UTJ ipsilateral to the
preovulatory follicle, close to ovulation time. In addition, the effects of low sperm dose,
inseminate volume, and sperm concentration were examined. The first-cycle pregnancy
rate served as the indicator of fertility.

2. Materials and methods

2.1. Stallions

Hanoverian breeding sires of proven normal fertility (n ¼ 26) were used in the
experiment. The stallions, belonging to the stud farm of the State of Lower Saxony at
Celle, Germany, were used in a commercial AI program that included fresh, shipped
cooled, and frozen–thawed semen. Since mare owners traditionally choose the stallion for
their mares, the number of stallions was not limited. Fresh semen was obtained from 21
and frozen–thawed semen from 17 stallions. The stallions were equally distributed within
the experimental groups. Their ages ranged from 4 to 18 years. During the experiment the
horses were kept indoors in box stalls bedded with straw. They were fed oats and hay three
times daily, and water was freely available.

2.2. Mares

A total of 331 reproductively normal-cycling Hanoverian mares were used in the

experiment during the 2002 breeding season (February–July). Mares to be inseminated
were stabled during estrus at the stud farm of the State of Lower Saxony at Celle, Germany.
The mares were kept in straw-bedded boxes in a separate stable and fed oats and hay three
times daily. Water was freely available.
918 H. Sieme et al. / Theriogenology 62 (2004) 915–928

Mares’ ages (3–21 years) were evenly distributed between treatment groups. The
average age was 8:6  4:1 years. The reproductive categories of foaling, maiden, and
barren were used. Since foaling and young maiden mares have a good fertility prognosis,
the group of 187 ‘‘normal mares’’ consisted of mares in foal (n ¼ 148, foaled in 2002 and
lactating throughout 2002) and young maiden mares (n ¼ 39, never bred, 3–4 years of
age). Mares with an abnormal reproductive history were considered to be ‘‘problem
mares’’ (n ¼ 85) when they had a history of barrenness (n ¼ 75, not pregnant after several
attempts throughout the 2001 season) or pregnancy failure (n ¼ 10, pregnancy failure
during or after the 2001 season; embryonic loss, n ¼ 3; abortion, n ¼ 3; stillbirth, n ¼ 4).
Ten mares had no anamnestic data and 14 had foaled earlier but had not been covered the
previous breeding season. These 24 mares were not grouped as either ‘‘normal’’ or
‘‘problem’’ mares.
Only clinically healthy mares with normal estrous cycles were eligible for participation,
each for a single heat. With the exception of the first estrus of foaling and young maiden
mares, uterine swabs were taken for bacteriological aerobic culture during the estrus
preceding the experiment. Mares showing positive cultures were excluded from the
experiment. Mares after normal foaling were inseminated either in foal heat (n ¼ 26
fresh semen, n ¼ 15 frozen–thawed semen) or in any subsequent heat (n ¼ 45 fresh semen,
n ¼ 62 frozen–thawed semen).

2.3. Experimental design

Mares were inseminated once per cycle with either fresh or frozen–thawed semen and
were randomly allotted into nine groups (21–48 mares per group) according to AI
technique. The mares were inseminated either by the conventional insemination technique
into the uterine body (ub), by transrectally guided deep intracornual insemination into the
uterine horn ipsilateral to the preovulatory follicle (ic), or by hysteroscopic insemination
onto the UTJ ipsilateral to the preovulatory follicle (hs). Fresh semen was used in volumes of
2 or 12 ml and in doses of 50
106 or 300
106 pms. The frozen–thawed semen dose
consisted of either a single 0.5 ml straw (Minitübe, Landshut, Germany) containing
100
106 total sperm or 8 straws. Extender was added to the semen volume of 4 ml to
make up the final 12 ml inseminate volume. The experimental design is presented in Table 1.
Mares were administered 1500 IU of hCG (Choriolutin1, Albrecht, Germany) iv at 10
a.m. when they were in estrus and follicle diameter was at least 40 mm. Transrectal
examinations and ultrasound scans (Aloka Echo Camera SSD-210 DX, Fa. Hellige, Ham-
burg, Germany) were carried out at 12 h intervals (10 a.m. and 10 p.m.) until detection of
ovulation. Mares were inseminated once per cycle, 24 h after hCG administration (10 a.m.)
with fresh semen or 30 h (4 p.m.) with frozen semen. An additional transrectal examination
was done immediately before the frozen semen AI at 30 h. Mares were removed from the
experiment if they failed to ovulate, had more than one ovulation, had already ovulated before
the examination 12 h (fresh semen AI) or 24 h (frozen–thawed semen) after hCG admin-
istration, or had not ovulated within 48 h. Thus, 35 mares were excluded from the original
331, leaving the data of 296 mares for analysis. Pregnancy examinations were performed by
ultrasonography 15–18 days post-ovulation at the stud farm. Possible embryonic loss rates
were recorded until day 43 of pregnancy by ultrasonography.
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 919

Table 1
Experimental design

Group Mares Insemination Semen Inseminate Sperm dose

(n) technique volume (ml) (
106)

ub-f 31 Uterine body Fresh 12 50 pms

ic-f 32 Deep intracornual Fresh 12 50 pms
hs-f 27 Hysteroscopic Fresh 12 50 pms
hs-f2 28 Hysteroscopic Fresh 2 50 pms
ub-f/300 34 Uterine body Fresh 12 300 pms
ub-ft 37 Uterine body Frozen–thawed 0.5 100 total
ic-ft 38 Deep intracornual Frozen–thawed 0.5 100 total
hs-ft 48 Hysteroscopic Frozen–thawed 0.5 100 total
ub-ft12/800 21 Uterine body Frozen–thawed 12 800 total
ub: conventional AI into the uterine body; ic: rectally guided deep intracornual insemination; hs: hysteroscopic
insemination; pms: progressively motile sperm; f: fresh; ft: frozen–thawed.

Clinical signs of post-breeding endometritis were detected by scanning the uterus for
intraluminal fluid accumulation 24 h after fresh semen AI and 18 h after frozen semen AI.
Intraluminal fluid collections greater than 10 mm in diameter were recorded using the
scoring described by Rasch et al. [31].

2.4. Preparation of semen

2.4.1. Fresh semen

Semen was collected for cooling once daily (6–7 a.m.) during the breeding season. Since
mares were inseminated with fresh semen 24 h after hCG (10 a.m.), the storage time
(3–4 h) of fresh semen was similar for all stallions and groups. The semen collections
took place on a phantom using an artificial vagina (Model Hanover, Minitüb, Landshut,
Germany), which was equipped with a sterile gauze filtration set in the collection device.
Immediately after collection, gel-free semen was evaluated for volume and spermatozoal
concentration by photometer (SpermaCueTM, Minitüb), and the total number of sperm per
ejaculate was calculated. The percentage of pms was estimated at 37 8C using a phase-
contrast microscope (Olympus CH-II, Olympus, Hamburg, Germany) with a stage heater
(HT 200, Minitüb). Semen was extended with modified skim milk extender (INRA 82, 32)
such that the final concentration was 25
106 pms/ml and then stored at 5 8C until use. To
inseminate mares with a small inseminate volume (group hs-f2), 2 ml of this semen was
taken to obtain 50
106 pms/AI dose. To mimic normal inseminate volumes, 10 ml of
extender (37 8C) was added immediately preceding insemination (groups ub-f, ic-f, hs-f).
To inseminate mares with a commercial sperm dose (group ub-f/300), 12 ml of semen
containing 300
106 pms was used. The percentage of pms was confirmed microscopi-
cally before AI.

2.4.2. Frozen semen

Frozen semen was processed in the routine freezing programme of the stud farm which
lasts from October to January. Semen collections were carried out every Monday,
920 H. Sieme et al. / Theriogenology 62 (2004) 915–928

Wednesday, and Friday. Semen was diluted in a modified skim milk extender [32] to a final
concentration of 50
106 sperm/ml and centrifuged for 10 min at 600
g. After decanta-
tion of the supernatant, sperm pellets were resuspended in the same skim milk extender
[32] which also contained 2% egg yolk and 2.5% glycerol. The final concentration was
200
106 sperm/ml. Semen was packaged into 0.5 ml straws (Minitüb) automatically
(MRS-1TM, Instruments Médécine Vétérinaire, L’Âigle, France), equilibrated for 60 min at
5 8C, and frozen automatically from 5 to 140 8C at 60 8C/min (MinidigitcoolTM,
Instruments Médécine Vétérinaire). The straws were thawed for 30 s at 37 8C in a water
bath. The frozen–thawed sperm dose contained 100
106 sperm with a post-thaw motility
above 35%; therefore, one 0.5-ml straw was thawed for a single insemination (groups: ub-
ft, ic-ft, hs-ft). After thawing of 8
0:5 ml straws, the resulting 4 ml was further diluted
with INRA 82 to obtain an inseminate volume of 12 ml (group ub-ft12/800).

2.5. Insemination techniques

All inseminations were carried out by the same two experienced technicians. Before
inseminations, the vulva was thoroughly cleaned with hygienic cotton paper. The intrau-
terine insemination catheter ‘‘IUI-pipette’’ (Minitüb) was used for uterine body and deep
intracornual inseminations. This is a flexible plastic AI pipette, 75 cm in length, and
suitable for fresh and frozen semen. The groups ub-f, ic-f, ub-f/300 and ub-ft12/800 were
inseminated using this catheter.
In combination with a stylet, the instrument also allows the transfer of frozen semen
packaged in 0.5-ml straws, similarly to the AI technique routinely used in cattle. After
thawing, the 0.5-ml straws were thoroughly dried, the seal-bearing end was cut with
scissors, and the straw was loaded into the AI pipette. If necessary, the pipette can remain in
the uterus and additional straws be reloaded in situ. The groups ub-ft and ic-ft were
inseminated using this technique.
In uterine body inseminations (ub-f, ub-f/300), the pipette described above was passed
through the cervix by transvaginal guidance using the finger to direct the pipette gently
through the cervix into the uterine body. The semen was deposited 3–5 cm behind the
uterine portion of the cervix.
In rectally guided deep intracornual inseminations (ic-f, ic-ft), the pipette was passed
through the cervix as described above. It was then gently pushed as far up the horn
ipsilateral to the preovulatory follicle as possible. The arm was removed from the vagina
and inserted into the rectum to grasp the uterine horn and manipulate the pipette gently to
the tip of the horn. The semen was injected as near to the tip of the horn as possible.
A flexible video-endoscope (Pentax EPM-3000, Videomed, München, Germany), 1.8 m
in length, with an outer diameter of 12 mm and a working channel of 2.8 mm, was used in
hysteroscopic inseminations (hs-f, hs-f2, hs-ft). The endoscope was preloaded introducing
a special polyethylene catheter (outer diameter: 1.52 mm; inner diameter: 0.86 mm;
Kleinfeldt, Gehrden, Germany) through the working channel. The endoscope, protected
by a glove that was withdrawn before the endoscope was passed through the cervix, was
guided transvaginally through the cervix and as far up the horn ipsilateral to the
preovulatory follicle as it easily went. The arm was then removed from the vagina and
inserted into the rectum to grasp the uterine horn and manipulate the endoscope to the tip of
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 921

the horn. Next, the assistant manipulating the operating unit of the endoscope started to
insufflate air until the papilla of the UTJ became visible. The catheter was extruded from
the working channel and directed to the papilla. Semen was injected slowly, drop by drop,
directly onto the papilla. The endoscope was withdrawn afterwards while simultaneously
evacuating the air.

2.6. Statistical analysis

All statistical comparisons were made using the SAS# statistics package (SAS Institute,
Cary, NC, USA). Three multivariate models were applied for testing the effects of
insemination technique, type of semen, and characteristics of mares and stallions on
pregnancy rates. In the first model, the factors of normal or problem mare, insemination
technique, and type of semen (fresh, frozen) were included. Possible interactions between
these three factors were analyzed using the procedure GENMOD and the logistic approach.
Significance was set at a P-value of 0.05. Due to the nested structure of some factors, two
new variables were created for the second model. These combined variables included
either the type of semen, inseminate volume, sperm dose, and sperm concentration, or all
mare-associated parameters (reproductive status of the mare, normal or problem mare, foal
heat). These factors made up the second model together with stallion characteristics as an
additional variable. Multivariate logistic model 3 was designed to evaluate the factor of
breeding foaling mares in foal heat or in a subsequent heat using insemination technique,
type of semen, and foal heat as the main factors.
Not only was the impact of these factors in general analyzed in the three models, but also
the estimated effects of different levels of these factors were tested against each other.
Confidence intervals for each level of each factor were calculated by retransformation of
the estimated mean to a normal scale. A 95% confidence interval was chosen, and the lower
and upper limits of these intervals were calculated together with the expectation values for
the most relevant effects.
Differences in pregnancy rates between treatment groups were analyzed by Chi-squared
(w2) test. For groups with 5 cycles, Fisher’s exact test was used. Statistical significance
was set at the 0.05 probability level.

3. Results

Because no significant differences in pregnancy rates were observed between the

different insemination volumes, sperm doses, or sperm concentrations (Table 2) or between
the different treatment groups (ub-f: 18/31, 58.1%; ic-f: 16/32, 50%; hs-f: 15/27, 55.6%;
hs-f2: 19/28, 67.9%; ub-f/300: 20/34, 58.8%; P > 0:05; ub-ft: 16/37, 43.3%; ic-ft: 17/38,
44.7%; hs-ft: 22/48, 45.8%; ub-ft: 12/800, 9/21, 42.9%; P > 0:05), effects of the different
insemination techniques were studied by combining data of the corresponding insemina-
tion groups: ub-f þ ub-f/300 into UB-f, hs-f þ hs-f2 into HS-f, and ub-ft þ ub-ft12/800
into UB-ft. In addition other groups were transformed automatically (ic-f to IC-f, ic-ft to
IC-ft, and hs-ft to HS-ft). The three insemination techniques had no significant effects on
post-breeding intrauterine fluid accumulation (UB-f: 7/65, 10.7%; IC-f: 3/32, 9.3%; HS-f:
922 H. Sieme et al. / Theriogenology 62 (2004) 915–928

Table 2
Effect of inseminate volume, sperm dose, and sperm concentration on pregnancy rates of mares using different
insemination techniques

Group Volume (ml) Pregnant/total %

ub-f, ic-f, hs-f, ub-f/300 12 69/124 55.7

hs-f2 2 19/28 67.9
ub-ft, ic-ft, hs-ft 0.5 55/123 44.7
ub-ft12/800 12 9/21 42.9
Sperm dose (
106 pms)
ub-f, ic-f, hs-f, hs-f2 50 68/118 57.6
ub-f/300 300 20/34 58.8
Sperm dose (
106 total)
ub-ft, ic-ft, hs-ft 100 55/123 44.7
ub-ft12/800 800 9/21 42.9
Sperm concentration (
106 pms/ml)
ub-f, ic-f, hs-f 4.16 49/90 54.4
hs-f2, ub-f/300 25 39/62 62.9
Sperm concentration (
106 total sperm/ml)
ub-ft, ic-ft, hs-ft 200 55/123 44.7
ub-ft12/800 66.6 9/21 42.9
pms: progressively motile sperm; f: fresh; ft: frozen–thawed sperm; ub-f: conventional AI into the uterine body
with 50
106 fresh pms/12 ml; ub-f/300: conventional AI into the uterine body with 300
106 fresh pms/
12 ml; ic-f: rectally guided deep intracornual AI with 50
106 fresh pms/12 ml; hs-f: hysteroscopic AI with
50
106 fresh pms/12 ml; hs-f2: hysteroscopic AI with 50
106 fresh pms/2 ml; ub-ft: conventional AI into the
uterine body with 100
106 total frozen–thawed sperm in one 0.5-ml straw; ub-ft12/800: conventional AI into
the uterine body with 800
106 total frozen–thawed sperm/12 ml; ic-ft: rectally guided deep intracornual AI
with 100
106 total frozen–thawed sperm in one 0.5-ml straw; hs-ft: hysteroscopic AI with 100
106 total
frozen–thawed sperm in one 0.5-ml straw.

3/55, 5.4%; P > 0:05; UB-ft: 3/58, 5.1%; IC-ft: 6/38, 15.7%; HS-ft: 7/48, 14.5%;
P > 0:05), pregnancy rates (UB-f: 38/65, 58.4%; IC-f: 16/32, 50%; HS-f: 34/55,
61.8%; P > 0:05; UB-ft: 25/58, 43.1%; IC-ft: 17/38, 44.7%; HS-ft: 22/48, 45.8%;
P > 0:05), or embryonic loss rates (UB-f: 4/38, 10.5%; IC-f: 0/16, 0%; HS-f: 3/34,
8.8%; P > 0:05; UB-ft: 4/25, 16%; IC-ft: 2/17, 11.7%; HS-ft: 2/22, 9.1%; P > 0:05) of
mares. Altogether 9.8% of mares exhibited ultrasonically visible intrauterine fluid, 8.5%
in fresh semen groups and 11.1% in frozen semen groups. A total of 9.8% of mares
experienced embryonic loss, 7.9% in fresh semen groups and 12.5% in frozen semen
groups.
Levels of probability for effects of the main factors (insemination technique, fresh versus
frozen semen, normal versus problem mares) on pregnancy rates of mares analyzed using
multivariate logistic model 1 were statistically significant only with respect to the semen
factor (P < 0:05) (Table 3). A significant effect was also seen when the combined influence
of insemination technique and category of mares as well as the combined influence of
insemination technique, type of semen, and category of mares were tested (P < 0:05).
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 923

Table 3
Analysis of factors influencing pregnancy rates of mares inseminated with reduced numbers of fresh and frozen–
thawed semen

Factors P-value

Multivariate logistic model 1

Insemination technique (UB, IC, HS) 0.557
Semen (fresh, frozen) 0.019*
Mares (normal, problem) 0.391
Semen
mares 0.544
Semen
insemination technique 0.476
Insemination technique
mares 0.039*
Insemination technique
semen
mares 0.020*
Multivariate logistic model 2
Combined sperm parameters: semen (fresh, frozen), volume, sperm dose and concentration 0.166
Combined mare parameters: status (maiden, barren, foaling), normal/problem, foal heat 0.110
Stallions 0.286
Insemination technique
combined sperm factors 0.597
Multivariate logistic model 3
Insemination technique (UB, IC, HS) 0.723
Semen (fresh, frozen) 0.006*
Foal heat 0.037*
Semen (fresh, frozen)
foal heat 0.221
Insemination technique (UB, IC, HS)
foal heat 0.181
Insemination technique (UB, IC, HS)
semen
foal heat 0.504
Statistical significance of variables including insemination technique, type of semen (fresh, frozen), and mare
characteristics (normal, problem) was tested in multivariate logistic model 1 (SAS, Proc GENMOD). In model 2,
variables tested included combined parameters for sperm (type of semen, volume, dose, concentration), mares’
reproductive history (status: maiden, barren, foaling; normal/problem mares; foal heat), and stallions. Model 3
tested the variables of insemination technique (UB, IC, HS), type of semen, and foal heat.
The symbol ‘
’ indicates interactions between variables; UB: conventional AI into the uterine body; IC: rectally
guided deep intracornual insemination; HS: hysteroscopic insemination; normal mares: maiden (3–4 years of
age) and mares in foal; problem mares: barren mares and mares with pregnancy failure.
*
P < 0:05.

Due to the nested structure of sperm parameters, a combined analysis (multivariate

logistic model 2) for the effects of these parameters (type of semen, volume, dose,
concentration) was carried out. No significant influences were discovered. Likewise,
combined mare parameters (reproductive status, normal/problem mares, foal heat) had
no effect. Pregnancy rates of mares inseminated with different stallions ranged from 0 to
100%. Considering only stallions with more than five observations, the stallion did not
count as a statistically relevant factor (Table 3).
Using a third statistical model, inseminating mares in foal heat had a significant effect on
pregnancy rates (P < 0:05) (Table 3). Fewer foaling mares became pregnant when
inseminated with fresh or frozen semen while in foal heat as compared with inseminations
in a subsequent heat (w2 test; fresh: 13/26, 50.0% versus 29/45, 64.4%; frozen: 3/15, 20.0%
versus 35/62, 56.4%; P < 0:05). Type of semen (fresh, frozen) was also found to be a
significant factor in this model. No interactive effect was present between foal heat, semen
type, and insemination technique (Table 3).
924 H. Sieme et al. / Theriogenology 62 (2004) 915–928

Table 4
Effects of insemination techniques using reduced numbers of fresh and frozen–thawed stallion sperm on
pregnancy rates of normal mares and mares with abnormal reproductive history, as analyzed by multivariate
logistic model 1 and w2 test

Group Mares w2 test Multivariate logistic analysis

Pregnant/total % Expectation value Confidence interval

Lower limit Upper limit

UB-f Normal 18/38 47.3 b 47.8 b 24.2 72.3

Problem 16/19 84.2 a 95.3 a 67.0 99.8
IC-f Normal 10/18 55.6 58.8 24.0 87.5
Problem 4/8 50.0 50.0 8.3 91.7
HS-f Normal 27/38 71.0 a 84.8 a 62.1 96.0
Problem 5/15 33.3 b 24.4 b 3.9 64.8
P
Fresh Normal 55/94 58.5
Semen Problem 25/42 59.5 x
UB-ft Normal 18/37 48.6 47.9 b 24.3 72.5
Problem 6/17 35.2 27.2 b 5.5 65.1
IC-ft Normal 12/25 48.0 43.4 b 16.6 73.8
Problem 5/13 38.5 31.9 b 5.6 74.1
HS-ft Normal 15/31 48.4 47.4 b 22.1 73.9
Problem 5/13 38.5 31.9 b 5.6 74.1
P
Frozen Normal 45/93 48.4
Semen Problem 16/43 37.2 y
Values with different letters (a, b) differ significantly (P < 0:05) within columns between groups; (x, y):
P < 0:05.
Normal mares: maiden (3–4 years of age) and mares in foal; problem mares: barren mares and mares with
pregnancy failure; pms: progressively motile sperm; f: fresh; ft: frozen–thawed sperm; UB-f: uterine body AI
with 50
106 or 300
106 fresh pms/12 ml; IC-f: rectally guided deep intracornual AI with 50
106 fresh
pms/12 ml; HS-f: hysteroscopic AI with 12 or 2 ml of semen containing 50
106 fresh pms; UB-ft: uterine body
AI with 100
106 total frozen–thawed sperm/0.5 ml or 800
106 /12 ml; IC-ft: rectally guided deep
intracornual AI with 100
106 total frozen–thawed sperm in one 0.5 ml straw; HS-ft: hysteroscopic AI with
100
106 total frozen–thawed sperm in one 0.5-ml straw; 95% confidence interval with lower and upper limits
calculated together with the expectation values.

Effects of insemination techniques (UB, IC, HS) on pregnancy rates of normal mares
and mares with abnormal reproductive history are summarized in Table 4 by a separate
analysis using multivariate logistic model 1 as well as w2 test. In problem mares, AI with
frozen semen yielded lower pregnancy rates than AI with fresh semen (P < 0:05),
whereas in normal mares there was no statistical difference. In normal mares, hystero-
scopic AI with fresh semen resulted in higher pregnancy rates than uterine body AI
(P < 0:05), but in problem mares the reverse was true (P < 0:05). Problem mares had
higher pregnancy rates than normal mares when inseminated into the uterine body with
fresh semen (P < 0:05), but the opposite effect was observed with hysteroscopic
insemination (P < 0:05).
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 925

4. Discussion

This study to our knowledge is the first report evaluating differences in pregnancy rates
between normal and problem mares using different insemination techniques. With fresh
semen, problem mares showed a significant decrease in pregnancy rate when inseminated
hysteroscopically as compared with routine insemination into the uterine body, but in
normal mares the effect was exactly the opposite. Uterine body insemination of problem
mares tended to yield higher pregnancy rates than rectally guided deep intracornual
insemination. Interestingly, pregnancy rates after uterine body insemination were higher in
problem mares than in normal mares. Thus, deep intrauterine insemination, either
hysteroscopic or rectally guided, cannot be recommended for stud farm practice to
increase fertility in problem mares. However, in normal mares, hysteroscopic insemination
seems to increase pregnancy rates when using low numbers of fresh sperm. Schiemann et al.
[33] examined eight healthy, diestrous mares 5 days after diagnostic hysteroscopy and
found that four of eight had pathogenic microbes. Furthermore, six mares showed
inflammation and marked eosinophilia in histological specimens of endometrial biopsies.
The authors recommended a follow-up treatment for mares that undergo hysteroscopy. It
has also been observed that the incidence of postbreeding endometritis after hysteroscopic
insemination is negligible [34]. Further studies are needed to determine the hygienic risk
associated with hysteroscopic inseminations in problem mares, as previously also sug-
gested by Squires et al. [24].
It was not surprising that pregnancy rate in foal heat was a statistically significant factor
since lowered foal heat pregnancy rates have been reported earlier [35,36]. However, no
interaction was present between foal heat and the main factors of semen characteristics and
insemination technique.
In our study, the lower pregnancy rates with frozen–thawed semen were accounted for by
‘‘problem’’ mares. However, frozen–thawed semen pregnancy rates of problem or normal
mares were not influenced by technique or site of insemination, sperm dose, or volume of
inseminate, suggesting that these factors are not as important as the fertility of the mare.
Our criteria for categorizing mares as barren (problem mares), foaling, and young maiden
(representing mares of normal fertility) are the same as those commonly used in many other
studies [3,5–7]. Vidament et al. [5] reported similar foaling rates for barren, foaling and
maiden mares following insemination with frozen–thawed semen. In another study, mare
status has, however, been shown to affect pregnancy outcome with frozen–thawed semen;
old maiden mares were the most problematic group in a multi-center study analyzing 2289
single inseminations in 1161 mares [7]. Moreover, pregnancy rates of older mares
inseminated with frozen–thawed semen are reduced [5–7].
Pregnancy rates of all mares inseminated with either fresh or frozen–thawed semen and
using three different insemination sites/techniques were not statistically different. Deep
intracornual low-dose insemination of fresh and frozen–thawed stallion semen seems to
lead to reasonable pregnancy rates, but whether intracornual AI is preferable to uterine
body AI is controversial. Morris et al. [27] inseminated mares with 25
106 frozen–
thawed pms and found hysteroscopic AI to hold no advantage over uterine body
insemination (9/14, 64% versus 9/12, 75%). However, when these authors used the same
technique and only 5
106 frozen–thawed pms, insemination on the UTJ resulted in
926 H. Sieme et al. / Theriogenology 62 (2004) 915–928

higher pregnancy rates than insemination into the uterine body (16/34, 47% versus 2/8,
25%). Furthermore, hysteroscopic insemination of 3
106 frozen–thawed pms in a 0.1-ml
volume resulted in better pregnancy rates than uterine body AI (16/34, 47% versus 2/14,
14%), but when the dose was increased to 14
106 pms in 0.5 ml, pregnancy rates were
almost equal (67% versus 64%) [23]. The advantage of hysteroscopic insemination can be
seen most clearly when using very low doses, i.e. 5
106 pms. For insemination with high
sperm numbers, uterine body AI seems to be the preferred site. Transrectal uterine horn AI
was shown to give lower pregnancy rates than uterine body AI (20% versus 50%) using
200
106 frozen–thawed sperm in a 0.5-ml volume [24]. Whether very low doses are
really necessary in stud farm practice is questionable, bearing in mind that normal stallions
are efficient sperm producers [37]. However, hysteroscopic AI is of great value for the
insemination of low numbers of fresh, stored and frozen–thawed sex-sorted sperm, as
reported by Lindsey et al. [15–17].
Uterine body insemination has usually been used as a control treatment, and surprisingly
good results have been reported when this conventional technique has been combined with
low semen doses [5,10,38]. In the present study, the low doses were about 10% of the
commonly recommended doses. Despite this, acceptable pregnancy rates were obtained
also by uterine body AI. Our findings and the results of others suggest that perhaps sperm
dose recommendations should be reevaluated. While large variations among individual
stallions exist, at least for fertile stallions producing good-quality semen with good
freezability, smaller doses might be sufficient. Reducing frozen semen doses would make
stallion semen freezing programs more efficient and economical. Popular stallions could
also have a larger book of mares if shipped semen were used.
Stallion was not found to be a statistically relevant factor. While in most studies that have
compared the effect of insemination techniques on pregnancy rates of mares the number of
stallions has been limited, our study was conducted with a large cohort of stallions
originating from a fertile stallion population. Therefore, extrapolation of the findings to a
wider population of stallions with normal fertility seems justified. Morris and Allen [34]
concluded that the use of hysteroscopic insemination did not improve results of infertile
stallions. Ismer [39], by contrast, observed increased pregnancy rates when mares were
inseminated hysteroscopically (37/68, 55.2%), as compared with conventional AI (3/8,
37.5%), into the uterine body with semen from one stallion that had shown asthenozoos-
permy and a history of reduced fertility. Applications of low-dose insemination techniques
for less fertile stallions have been discussed in the literature, but their usefulness remains
debatable. Further research is required to determine which sperm qualities can be
compensated for by the techniques.
In summary, neither insemination technique, mare characteristics (normal, problem),
stallions, nor sperm parameters (inseminate volume, sperm dose, sperm concentration)
were significant factors in the present study. Type of semen (fresh, frozen), breeding mares
during foal heat, and the interaction between insemination technique and semen and mare
characteristics were, however, found to be significant factors (P < 0:05). Timed single
inseminations with reduced sperm doses were as successful as conventionally recom-
mended doses, irrespective of the insemination technique used, suggesting that commercial
doses of fresh or frozen–thawed semen from fertile stallions could be lowered without
risking a decrease in fertility at least when the inseminations are carried out close to
 H. Sieme et al. / Theriogenology 62 (2004) 915–928 927

ovulation. In problem mares, frozen semen AI resulted in significantly lower pregnancy

rates than fresh semen AI, but they were not influenced by insemination technique. Deep
intracornual insemination is not an optimal technique in problem mares. In normal mares,
the highest pregnancy rates can be expected by hysteroscopic insemination.

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with low numbers of either unsexed or sexed spermatozoa. Theriogenology 2000;53:1333–44.
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the mare—uterine contamination and endometrial reaction. Pferdeheilkunde 2001;6:557–64.
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[38] Nie GJ, Wenzel JGW, Johnson KE. Comparison of pregnancy outcome in mares among methods used to
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 animals
Review
Factors Influencing the Popularity of Artificial
Insemination of Mares in Europe †
Alicja Kowalczyk 1, * , Ewa Czerniawska-Piatkowska
˛ 2 and Marian Kuczaj 3
1 Department of Environment, Animal Hygiene and Welfare, Wrocław University Of Environmental and Life
Sciences, Chełmońskiego 38C, 51-630 Wrocław, Poland
2 Department of Ruminant Science, West Pomeranian University of Technology, Klemensa Janickiego 29,
71-270 Szczecin, Poland
3 Institute of Animal Breeding, Wrocław University Of Environmental and Life Sciences, Chełmońskiego 38C,
51-630 Wrocław, Poland
* Correspondence: alicja.kowalczyk@upwr.edu.pl
† Abbreviated Title: Popularity of Stallion Sperm Production in Europe.




Received: 27 May 2019; Accepted: 18 July 2019; Published: 19 July 2019


Simple Summary: The popularity of mare insemination as an element affecting the dynamic growth
of breeding progress among horses in Europe is subject to various fluctuations. The success of this
method of reproduction should be considered first of all in terms of the quality of semen available
on the market, the types of semen storage technology, the profitability of its use resulting from the
effectiveness of insemination with the selected type of semen, and factors affecting the success of
artificial insemination. The purpose of this work was to present the factors determining the popularity
of artificial insemination of mares in Europe. The presented statistics show that the popularity of
the use of chilled semen has gradually increased in the group of sport mares, while in the group
of breeding mares, the popularity of frozen semen has increased. In the remaining group of mares
(not classified as sport or breeding), insemination with chilled semen was dominant. To talk about
the success of artificial insemination of horses in Europe, it is necessary to look thoroughly at these
aspects that affect the popularity of this reproduction biotechnology, and in particular to improve the
quality of insemination doses.

Abstract: The purpose of this review was to analyze factors affecting the popularity of artificial
insemination of mares in Europe in the context of sperm quality. Taking into account the prices of
stallion semen on the world market, efficiency is important for the profitability of its use in artificial
insemination programs in Europe. To increase the efficiency of a semen insemination facility, it is
necessary to correctly and objectively assess the quality of semen. The available range of tools allows
an effective evaluation of the potential fertility of a stallion. For several years, artificial insemination
programs in Europe have been gaining popularity. However, the frequency of chilled or frozen semen
use is still quite low. This is mainly due to the common, negative opinion about the effectiveness of the
use of packaged insemination doses as opposed to natural insemination. Unfortunately, the quality
of the semen offered often deviates from expectations, which results in unsatisfactory (and therefore
unprofitable) pregnancy rates. This review presents the popularity structure of chilled and frozen
semen use in European horse breeding as well as the current state of research on the effectiveness of
semen production technology. It is shown that the popularity of using chilled semen in the artificial
insemination of mares in Europe has been gradually increasing in the group of sport mares, while
in the group of breeding mares, in recent years, frozen semen has been gaining popularity. In the
remaining group of mares (not classified as sport or breeding), insemination with chilled semen has
been dominant.

Keywords: equine; insemination; semen; stallion

Animals 2019, 9, 460; doi:10.3390/ani9070460 www.mdpi.com/journal/animals

Animals 2019, 9, 460 2 of 8

1. Introduction
Horse breeding in Europe is mainly based on the selection of horses in terms of sport and breeding
performance. To a much lesser extent, horses are selected for recreational use. The basic assumption
of horse reproduction in Europe is profitability, which results from the sale of a horse or its lease.
In Europe, the selection of horses for breeding is made based on the pedigree, sporting results, and
temperament of the animal concerned. The decisive factor when choosing a sire is the price of its semen.
Artificial insemination is widely used in modern animal reproduction [1], especially when
genetic improvement is taken into account. The percentage of foals coming into the world as a
result of insemination with chilled or frozen semen has reached about 90% [2]; in Europe, it is only
45% [3]. Methods of semen freezing, the composition of extenders for stallion sperm storage (necessary
to maintain the integrity of cytoplasmic membranes and fertilizing potential), insemination time,
insemination site, and the optimal insemination dose are the factors contributing to the variability
of pregnancy indices [4]. To minimize this variability, scientists and practitioners try to develop
optimal, standardized protocols both for sperm freezing and for insemination. Research is also aimed
at predicting the fertility of spermatozoa subjected to freezing and thawing. In particular, the attempts
are focused on the identification of specific molecular markers that can potentially be highly correlated
with fertility, and thus can increase the susceptibility to freezing and the effectiveness of artificial
insemination with stallion semen. The aim of this work is to analyze factors affecting the popularity of
different types of stallion sperm (chilled and frozen) in Europe.

The Structure of Chilled and Frozen Semen Use for Artificial Insemination in Europe.
As mentioned in the introduction, mares artificially inseminated in Europe constitute fewer
than half of those intended for reproduction. Table 1 presents data concerning the structure of mare
insemination in Europe in the period 2013–2017 (data come from insemination stations; they were
collected and archived based on private summaries from three leading breeding stations in northern
France (N = 986), south-western Poland(N = 554), and eastern Germany (N = 940)).

Table 1. The structure of mare insemination in Europe, including the division into their type of use
(N = 2480), developed based on reports from European insemination stations.

Mares
Years
Sport Breeding Other
2013 49% 37% 14%
2014 53% 40% 7%
2015 58% 33% 9%
2016 61% 37% 2%
2017 67% 29% 4%

Artificial insemination is used predominantly in sport mares and has been increasingly popular
since 2013. In 2017, sport mares accounted for 67% of all mares inseminated at the breeding stations.
In the case of breeding mares, a slight decrease in the use of artificial insemination as the main breeding
technique has been observed. The percentage of artificially inseminated mares decreased from 37%
in 2013 to 29% in 2017. No significant variability in the frequency of artificial insemination among
breeding mares has been observed in particular years. The other mares (not classified as sport or
typically breeding ones) constitute a small percentage of all artificially inseminated mares, and the
popularity of this kind of insemination among this group has been decreasing significantly since 2013.
Figures 1–3 show the popularity of semen used in mares breeding in Europe from 2013 to 2017,
taking into account the type of semen used for this purpose (chilled, frozen).
Animals 2019, 9, 460 3 of 8
Animals 2019, 9, x FOR PEER REVIEW 3 of 8
Animals 2019, 9, x FOR PEER REVIEW 3 of 8

Sport mares
Sport mares
70% 66%
62% 66%
70%
60% 55% 62% 56%
53%
60% 55% 56%
47% 53% 45% 44%
50%
47% 45% 44%
50% 38%
40% 38% 34%
40% 34%
30%
30%
20%
20%
10%
10%
0%
0% 2013 2014 2015 2016 2017
2013 2014 2015 2016 2017
Chilled Frozen
Chilled Frozen

Figure
Figure1. 1.The
Thestructure
structure of of insemination
inseminationofof sport
sport mares
mares in Europe,
in Europe, including
including the division
the division into theinto
typethe
Figure
type
of of
semen 1. The
semen structure
used
used forfor of insemination
artificial
artificial of sport
insemination
insemination (N(N = 2480),
= mares
2480), indeveloped
Europe, including
developed onthe
based
based on division
reports
reports intoEuropean
from
from the type
European
of semen used
insemination
insemination for artificial insemination (N = 2480), developed based on reports from European
stations.
stations.
insemination stations.
In In
2013
2013and
and2014,
2014, the popularity
popularityofofusing
using chilled
chilled semen
semen was was
lowerlower than frozen,
than frozen, while2015
while from from
2015 Inpopularity
its its 2013 andbegan
popularity 2014, the
began popularity
to to
grow
grow of using chilled
dynamically
dynamically (by(by semen
about
about15%was
15% lower
compared than
compared tofrozen,
topreviouswhile
previous from 2015
years).
years).This
This
its popularity
popularity
popularity began
reached62%
reached to grow dynamically
62%ofofinseminated
inseminated sport (by
sport mares about
mares and 15%
and was
was24%compared
24%higher to
higherthan previous
thanininthe years).
case
the of of
case This
frozen
frozen
popularity
semen. reached 62%
semen.InInsubsequent
subsequent of inseminated
years
years (2016
(2016and sport
2017),
and mares
it remained
2017), and
at was
it remained at24%
a similar higherstill
level,
a similar than in thedominating
dominating
level, still case of frozen
over over
semen.
frozen semen.In subsequent years (2016 and 2017), it remained at a similar level, still dominating over frozen
semen.

Breeding mares
Breeding mares
70% 64%
70% 61%
64%
60% 55% 61%
52%
60% 55% 45% 48% 49% 51%
52%
50% 45% 39%
48% 49% 51%
50% 36%
40% 39% 36%
40%
30%
30%
20%
20%
10%
10%
0%
0% 2013 2014 2015 2016 2017
2013 2014 2015 2016 2017
Chilled Frozen
Chilled Frozen

Figure 2. The structure of insemination of breeding mares in Europe, including the division into the
Figure
type 2.
Figure The
of 2. Thestructure
semen used forof
structure ofinsemination
insemination
artificial of
of breeding
breeding
insemination mares
mares
(N = 2480), inEurope,
in Europe,
developed including
including
based the
the
on reports division
division
from into
into thethe
European
type
typeof semen
of semen used for
used
insemination stations. forartificial
artificialinsemination
insemination (N
(N =
= 2480),
2480), developed
developed based
based on
on reports
reports from
from European
European
insemination
insemination stations.
stations.
 Animals 2019, 9, x FOR PEER REVIEW 4 of 8

Initially, in the period of 2013–2015, the use of chilled semen in the artificial insemination of
breeding mares significantly prevailed over frozen, representing 55% (2013), 61% (2014) and 64%
(2015) of all breeding mares submitted to insemination in the European insemination centers.
Animals 2019, 9, 460 4 of 8
However, since 2016, there has been a significant increase in the frequency of the use of frozen semen,
reaching a similar level as the use of chilled semen.

Other mares

90% 84%
80%
80% 75%
71%
70% 65%

60%
50%
35%
40% 29%
25%
30% 16% 20%
20%
10%
0%
2013 2014 2015 2016 2017

Chilled Frozen

Figure
Figure3. 3.The
Thestructure
structure ofof insemination
inseminationofof other
other mares
mares in Europe,
in Europe, including
including the division
the division into theinto
typethe
type
of of semen
semen used
used forfor artificial
artificial insemination
insemination (N=
(N= 2480),
2480), developed
developed based
based on on reports
reports from
from European
European
insemination
insemination stations.
stations.

Initially, in themares,
In the other period theofuse
2013–2015, the useconstitutes
of frozen semen of chilleda semen in the artificial
small percentage, whichinsemination
is on average of
breeding
about 25% mares significantly
of all mares in this prevailed over2015,
group. Since frozen,
therepresenting
use of chilled55% (2013),
semen 61% (2014)more
is significantly and 64% (2015)
popular
of than
all breeding
frozen, mares
although submitted
in recenttoyears
insemination
(2015, 65%;in the European
2016, 71%; 2017,insemination centers. However,
75%) its popularity since
has slightly
decreased.
2016, there has been a significant increase in the frequency of the use of frozen semen, reaching a
similar level as the use of chilled semen.
2. Semen Evaluation
In the other mares, the use of frozen semen constitutes a small percentage, which is on average
about 25% of all mares
In Europe, only a in this
few group. Since
laboratories 2015, semen
in stallion the useproduction
of chilled stations
semen is significantly
routinely more
use flow
popular
cytometry to examine sperm quality. The other stations use the conventional method, i.e., thehas
than frozen, although in recent years (2015, 65%; 2016, 71%; 2017, 75%) its popularity
slightly decreased.
determination of the percentage of pregnancies achieved among mated/inseminated mares. The use
of cytometric methods to improve the quality of biological value of stallion ejaculate evaluation is not
2. aSemen
routine Evaluation
procedure in Europe, and the data obtained in the area of individual stallion fertilization
effectiveness
In Europe,are onlyquite limited.
a few As demonstrated
laboratories in the
in stallion latestproduction
semen research, it is possibleroutinely
stations to obtain use
a high
flow
cytometry to examine sperm quality. The other stations use the conventional method, i.e.,ofthe
level of stallion fertility prediction by (partly) introducing cytometric analysis as the basic method
semen evaluation and by establishing a new protocol for semen evaluation [5]. By combining
determination of the percentage of pregnancies achieved among mated/inseminated mares. The use of
microscopic observations, computerized motility analysis and flow cytometry, objective and practical
cytometric methods to improve the quality of biological value of stallion ejaculate evaluation is not
tools to estimate a stallion’s fertility can be provided. The evaluation of stallion semen before its
a routine procedure in Europe, and the data obtained in the area of individual stallion fertilization
application in breeding and artificial insemination (AI) is crucial in the case of breeding horses.
effectiveness are quite limited. As demonstrated in the latest research, it is possible to obtain a high
Failure to eliminate poor quality ejaculates may result in poor fertility rates, and this is the most
level of stallion fertility prediction by (partly) introducing cytometric analysis as the basic method
important issue in the evaluation of a stallion’s reproductive performance. Gottschalk et al. [6]
of indicates
semen evaluation and bybetween
the relationship establishing a newofprotocol
the fertility stallionsfor
andsemen
semenevaluation
traits. The [5]. By of
indices combining
semen
microscopic observations, computerized motility analysis and flow cytometry,
quality, including progressive movement, complete sperm motility and morphology, may explain objective and practical
tools
some to fluctuations
estimate a stallion’s fertility
in a stallion’s can [7–9].
fertility be provided. Theofevaluation
The fertility of stallion
stallions should semen before
be measured by the its
application in breeding and artificial insemination (AI)
degree of pregnancies per cycle [2,10,11] or non-return rate [12,13].is crucial in the case of breeding horses. Failure
to eliminate poor quality ejaculates may result in poor fertility rates, and this is the most important
issue in the evaluation of a stallion’s reproductive performance. Gottschalk et al. [6] indicates the
relationship between the fertility of stallions and semen traits. The indices of semen quality, including
progressive movement, complete sperm motility and morphology, may explain some fluctuations in a
stallion’s fertility [7–9]. The fertility of stallions should be measured by the degree of pregnancies per
cycle [2,10,11] or non-return rate [12,13].
Animals 2019, 9, 460 5 of 8

3. Semen Storage
Cryopreservation is currently the only possible method of sperm storage for an indefinite period.
However, the cryopreservation and thawing processes reduce sperm viability and motility in all
studied farm animal species, including horses [14–16]. Also, there is evidence that cryopreservation
leads to DNA defragmentation, which is important in terms of fertilization and normal embryonic
development [17]. Many of the harmful effects resulting from cryopreservation can be attributed to
osmotic stress. The formation of extracellular ice crystals starts during cooling below the temperature of
0 ◦ C. This, in turn, causes a large increase in osmolarity, which exposes sperm cells to extreme osmotic
stress [18]. Also, cryoprotectants produce hyperosmotic cryodiluent, which causes cell dehydration
through osmosis. Since dehydration is essential for maintaining sperm viability after thawing, extreme
hyperosmolarity induces cellular stress (water flows through the sperm membrane to the water
channels, attempting to balance osmolarity) [19].The result of these osmotic stressors is damage to
the cytoplasmic membranes [20], DNA damage [21] and the production of reactive oxygen species
(ROS) [22]. However, besides the possibility of long-term storage of stallion semen, chilled semen is
also used in Europe. In this case, the semen is usually processed using commercial passive cooling
devices that slowly cool the diluted semen to about 5 ◦ C, a temperature that is low enough to limit the
cell metabolism to a sufficient degree to maintain the function of the semen up to about 72 h after its
collection. However, stallion spermatozoa are much more susceptible to cold shock than the sperm of
other species. As in the case of cryopreservation, there are numerous unexplained differences between
stallions in the suitability of their semen for storage at low temperatures [23].

4. Time of Insemination
Bearing in mind the superior breeding goal (a horse with high sport value), European horse
breeders, from an economic point of view, desire the horses to be born in early spring. This is because
horses competing in sport arenas born in the same year, but at intervals of a few months, will be
characterized by a significantly different degree of training and differences in body composition and
development. Considering that a sport horse is supposed to achieve the desired results in a selected
competition, the season of birth is of great importance in the context of acquired skills and experience.
The time when a foal is born is, therefore, an important economic factor. Another important timing
factor for breeding profitability is insemination time, since both too early and too late sperm deposition
in the female reproductive tract is not only a reason for losing valuable time, but also a reason for losing
funds invested in the purchase of semen and the preparation of mare insemination. Insemination with
cryopreserved semen should take place at a time similar to ovulation; specific protocols developed to
coordinate the insemination time with ovulation induction yielded promising results. Samper showed
that most mares (94%) ovulate between 36 and 42 h after ovulation induction using human chorionic
gonadotrophin (hCG) or deslorelin (if the mare is in oestrus, and the dominant preovulatory follicle
and endometrial oedema are present at the time) [24]. There are, however, conflicting theories about the
optimal time of insemination in relation to ovulation itself. Many research laboratories have examined
pregnancy indices concerning mare insemination before and after ovulation. Some researchers argue
for the insemination of mares both before and after ovulation. They recommend the insemination
of mares at 24 and 40 h after ovulation induction, while others recommend it at 36 h and then again
between 42 and 44 h after ovulation induction. For both schemes, it is assumed that ovulation occurs
between two inseminations. Other researchers say that only one insemination is needed for acceptable
pregnancy rates. Barbacini et al. [25] evaluated insemination protocols and found no difference in
pregnancy rates between mares inseminated at 24 and 40 h after hCG administration (46%), (ovulation
likely occurred between 36 and 42 h) and the pregnancy rate in mares which were inseminated only
once after ovulation induction (47%). Single insemination after ovulation provided similar pregnancy
rates per cycle, as reported by Hemberg et al. [26] (45.4%) and Metcalf [27] (47%). From an economic
point of view, the most profitable option for the breeder is the induction and monitoring of ovulation,
insemination at the time most similar to ovulation, and simultaneous use of the smallest possible
Animals 2019, 9, 460 6 of 8

number of semen doses. Considering that the majority of stallion stations in Europe offer single
portions of semen (one straw of 0.5 mL of frozen semen or one vial of 20 mL of chilled semen) in the
price range from €450 to €3500, it becomes clear that the less semen used per effective insemination,
the lower the cost of one pregnancy.

5. Reduction in the Number of Spermatozoa

A good effectiveness of fertilization can be achieved by using frozen/thawed semen with reduced
sperm concentration, using insemination near the follicle. Recently, research has focused on deep
insemination (to uterine horns) or hysteroscopy. Although hysteroscopic insemination was originally
aimed at improving pregnancy rates in mares inseminated by low fertility stallions [28], this technique
did not yield the desired effects in this case [29]. Indicators of pregnancy after hysteroscopic or
deep insemination using a small concentration of sperm are very diverse for frozen/thawed semen.
Lindsey et al. [30] found a 37.5% pregnancy rate in semen inseminated with sperm containing 15,106
active frozen/thawed spermatozoa inseminated with hysteroscopy, while Morris et al. [31] found a
higher prevalence rate of 64.3% (9/14) after insemination with a dose of 14,106 motile, frozen/thawed
sperm. The author obtained the same prevalence of pregnancy when mares were inseminated with
a dose of 14,106 motile, frozen/thawed sperm to the uterine body (66.7%, 8/12). Petersen et al. [32]
reported 64% (7/11) pregnancies among mares inseminated with a dose of 50,106 frozen/thawed,
progressively motile spermatozoa at 12 and 24 h after hCG administration, using deep insemination to
the uterine horn. When the same mares were inseminated with a dose of 500,106 sperm cells to the
uterus, only 4/11 mares became pregnant (37%).

6. Conclusion
The analysis of stallion sperm is an important part of reproductive performance evaluation.
The results of semen evaluation cannot be interpreted without a thorough knowledge of mares and
of the management effects of the herd that could play a role in or affect the potential fertility of the
stallion. Such an evaluation should be carried out using modern tools such as flow cytometry, allowing
objective, fast and simultaneous analysis of many quality parameters while analyzing a large number
of spermatozoa. Bearing in mind the development of artificial insemination in Europe, it is important
to popularize preserved semen by improving its quality and its effectiveness of use in insemination.
The popularity of using chilled semen in the artificial insemination of mares in Europe has been
gradually increasing in the group of sport mares, while in the group of breeding mares, in recent years,
frozen semen has been gaining popularity. In the remaining group of mares (not classified as sport or
breeding), insemination with chilled semen has been dominant. Likely, the popularity of the type of
semen used for insemination is primarily determined by the effectiveness of insemination with the
chosen type of dose (chilled or frozen) and the biological quality of the ejaculate stored in a liquid or
frozen state, which influences this effectiveness.
The main purpose of an artificial insemination program is to determine the most effective use of
semen without lowering the rate of pregnancy obtained. Insufficient sperm quality, whether chilled or
frozen, directly affects the popularity of its use, affecting breeders’ profit levels (not only for the owner
of the mares, but also for the owner of the stallion/producer of the semen).

Author Contributions: We declare that all authors made substantial contributions to this manuscript. A.K. in
conducting the experiment, in the conception and design of the study, and in establishing the methodology. M.K.
in the collection and assembly of data. A.K. and E.C.-P. in the analysis and interpretation of results and in the
preparation of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Animals 2019, 9, 460 7 of 8

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