Theriogenology 59 (2003) 11231130

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration
Gianluca Negliaa, Bianca Gasparrinia, Viviana Caracciolo di Brienzaa, Rossella Di Paloa, Giuseppe Campanilea, Giorgio Antonio Presicceb, Luigi Zicarellia,*
a

Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, Federico II University, Via Delpino 1, 80137 Napoli, Italy b Centro Sperimentale per la Zootecnia, Rome, Italy Received 28 August 2001; accepted 15 April 2002

Abstract This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP). Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage. Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P < 0:05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P < 0:05) than Group B. Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P < 0:05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo. In conclusion, in buffalo species the source of oocytes signicantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes. A higher overall IVEP efciency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: In vitro embryo production; Ovum pick-up; Buffalo; Bovine
* Corresponding author. Tel.: 39-81-4421928; fax: 39-81-292981. E-mail address: zicarell@unina.it (L. Zicarelli).

0093-691X/02/$ see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 7 0 - 6

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1. Introduction In vitro embryo production procedures are applied worldwide with different goals for a variety of livestock species, exotic, wild and endangered animals [15]. The possibility of obtaining embryos from in vitro technology has drawn the attention of animal scientists and entrepreneurial groups, especially when a non invasive procedure for recovering oocytes from antral follicles in live animals became a practical and effective approach [611]. Collection of oocytes by means of ultrasound guided follicular aspiration is routinely performed with success in large prepuberal and adult ruminant species under various physiological and pathological conditions [1216]. Because of its high milk production, the Mediterranean Italian buffalo is in high demand around the world, as well as important for the local economy. Special efforts are needed to rapidly propagate superior males and females. The application of OPU technology, together with multistep embryo production in vitro, could be a way of meeting this need. Traditional reproductive technologies are currently utilized in this species, but some inherent physiological features such as reduced expression of behavioral estrus [17,18], a lower number of follicles compared to the bovine species [19] and possibly a reduced responsiveness to hormonal stimulation [2022] collectively render buffalo somewhat less efcient reproductively compared to the bovine species. The application of OPU technology to buffalo, together with an improvement of the multistep process of in vitro embryo production, can enhance genetic progression through the maternal lineage, thus overcoming the reduced level of efciency linked to more traditional approaches [23,24]. However, in vitro embryo production in the buffalo species has always been associated with a lower efciency compared to the bovine species [24,25], although recently some encouraging improvements have been reported [26]. The aim of this study was to elucidate possible differences between in vitro embryo output from the bovine and buffalo species, using oocytes originating from abattoir ovaries (bovine and buffalo) and oocytes collected by transvaginal follicular aspiration (buffalo), under the same laboratory conditions. 2. Materials and methods 2.1. Oocyte source Eight non lactating pluriparous buffalo cows, at the end of their economic life, underwent repeated transvaginal follicular aspiration for a period of 6 months, from March until September. All animals were barnhoused and under controlled nutrition. Three hundred and thirty-eight cumulus oocyte complexes (COCs) were recovered. Ovum pick up was usually carried out twice weekly on the farm, with oocyte search performed immediately after follicular aspiration. For direct comparison, buffalo and cow ovaries were collected from local slaughterhouses once a week (17 sessions) during the same weeks as OPU (34 sessions), and brought to the laboratory within 4 h from their excision. Ovaries belonged to a random pool of dairy pluriparous animals slaughtered at the end of their economic life. From these ovaries a total of 1127 buffalo and 1457 bovine COCs were collected. In the

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present study, only the OPU sessions carried out when bovine and buffalo ovaries from slaughterhouse were also available, were included. 2.2. Transvaginal follicular aspiration Ovum pick up equipment consisted of a portable ultrasound unit (SSD-500, Aloka Co., Tokyo) with a 5.0 MHz sector scanner mounted on a properly designed vaginal support, equipped with a needle guide. The 50 cm long needle (17 gauge) was connected to a regulated vacuum pump (K-MAR-5100, Cook IVF Co., Australia) which draws the samples into a 15 ml sterile tube. Animals were restrained in a squeeze chute and given an epidural administration of 46 ml of 2% lidocaine (Gellini, Italy). A vacuum pressure of 40 mm Hg was constantly maintained and the aspiration line was continuously rinsed with 25 mM hepes buffered TCM 199 supplemented with 100 USP units/ml of heparin during follicular aspiration. All visible antral follicles were punctured. 2.3. Oocyte processing Media and chemicals used for gamete and embryo processing were purchased from Sigma (Sigma, Milano, Italy). The COCs recovered by ultrasound guided aspiration were searched immediately after ltering the aspirated follicular uid and aspiration medium and graded: COCs characterized by uniform cytoplasmic appearance and enclosed within 3 (Grade B) or more (Grade A) layers of viable compact granulosa cells were considered suitable for in vitro production. Grade C oocytes (COCs characterized by less than three layers of granulosa cells or partially denuded) were included for further processing but cultured in separated drops. The oocytes were washed twice in Hepes-buffered TCM 199 (H 199) with 10% FCS and then allocated in the same medium supplemented with 0.5 mg/ml FSH, 5 mg/ml LH, 1 mg/ ml estradiol and 50 mM cysteamine. Processed oocytes were stored in 15 ml Falcon tubes in a portable incubator at 39 8C and moved to the lab within 46 h, where they were transferred into 50 ml droplets under mineral oil of nal maturation medium consisting of bicarbonate-buffered TCM 199 (B 199) with 10% FCS, 0.5 mg/ml FSH, 5 mg/ml LH, 1 mg/ ml estradiol and 50 mM cysteamine. Abattoir ovaries were stored in saline in a portable incubator at 3035 8C and moved to the same lab within 34 h for further processing. Briey, COCs were recovered from small to medium size follicles (28 mm), washed twice in H 199 and once in nal maturation medium previously dened. Both types of processed abattoir-derived COCs were then placed for in vitro maturation into 50 ml droplets (10 COCs per droplet) of B 199 and hormones with the addition of 50 mM/ml cysteamine for buffalo oocytes [26] and 100 mM/ ml for bovine oocytes [27]. In this study, Grade A and B oocytes were placed into maturation droplets covered by mineral oil and incubated at 38.5 8C (buffalo) and 39 8C (bovine) for 2224 h under controlled gas atmosphere consisting of 5% CO2 in humidied air. The length of maturation time for OPU-derived oocytes is the same as for abattoir oocytes, but maturation is considered to start from the exposure of the oocytes to the hormones immediately after recovery. Therefore, time into controlled atmosphere for this source of oocytes is reduced when compared to abattoir-derived oocytes.

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2.4. Fertilization and embryo culture In both species spermatozoa were prepared from frozen-thawed semen, obtained from commercial standard bulls that were previously tested for IVF in our laboratory. In our experience the optimal sperm concentration differed for bovine and buffalo bulls. Sperm was treated by swim-up procedure in a modied version of Ham's medium for 1 h. The pellet obtained after centrifugation of supernatant was resuspended to a nal concentration of 1 106 /ml (bovine) and 2 106 /ml (buffalo) in the fertilization medium, a modied TALP supplemented with 0.2 mM/ml penicillamine, 0.1 mM/ml hypotaurine and 0.01 mM/ml heparin. Fertilizing droplets (5 COCs/50 ml droplet) covered by mineral oil were incubated under the same gas atmosphere as for in vitro maturation. Sperm oocytes co-incubation lasted 2022 h. Presumptive zygotes were moved into 20 ml droplets (10 per droplet) of a Hepes-buffered version of synthetic oviduct uid (SOF) medium [28] with the addition of essential and non essential amino acids overlaid with mineral oil in a modulation chamber (Forma Scientic, USA), with a gas atmosphere of 5% CO2, 7% O2 and 88% N2 [28]. The chamber was gassed at the time of incubation (5 min were allowed for gassing) and at Day 5, when embryos were transferred into fresh medium, to avoid toxicity due to high levels of NH4. Cleavage was assessed on the same day in order to reduce changes of gas atmosphere during early embryo development within the chamber. Final embryo output (Tm and Bl) was evaluated at Day 7 of culture. 2.5. Statistical analysis Chi-square analysis was used for data evaluation and statistical interpretation. Differences were considered signicant at the 5% level. 3. Results Within Grade A B oocytes, considered suitable for IVEP, the incidence of Grade A oocytes was 45, 55 and 80% in Groups A, B and C, respectively. A very low proportion of Grade C OPU-derived buffalo oocytes developed to blastocyst stage (3.3%). Results reported in Table 1 include only Grade A and B oocytes for all the groups considered. A high individual variability was characteristic of buffaloes subjected to OPU, as reected by higher S.D. (Table 1). A higher embryo yield was obtained in Group A compared with Group B within buffalo species (Table 1). An overall better IVEP efciency was recorded in Group C compared with Group B (Table 1). Furthermore a higher proportion of morula to total embryos was observed in Group B compared with Groups C and A, as shown in Table 2. This is conrmed by the nding that the numbers of Tm Bl, and of Bl, calculated as a percentage of total COCs, were not different within Group A (33.1% versus 29.7%) whereas differences were found within Group B (25.7% versus 19.9%; P < 0:01) and Group C (33.7% versus 29.7%; P < 0:05). Similar results were observed also when the numbers of Tm Bl, and of Bl were calculated as a proportion of total cleaved oocytes.

G. Neglia et al. / Theriogenology 59 (2003) 11231130 Table 1 Embryo developmental parameters (mean S:D:) from the two different sources of species and oocytes Oocytes (n) Cleavage (%) Tm Bl/tot1 (%) Tm Bl/clv2 (%) Bl/tot3 (%) Group A 338 Group B 1127 Group C 1457 66.4 23.7a 64.8 9.6a 83.9 7.6b 33.1 19.0a 25 7 4.2b 33.7 4.3a 52.2 24.0a 40.1 6.6b 40.3 4.9b

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Bl/clv4 (%)

29.7 19.5a 47.6 26.4a 19.9 4.2b 31.3 7.9b 29.7 4.5a 35.6 5.5c

Columns with different superscripts (ac) differ (P < 0:05). 1 Tight morula and blastocysts/total oocytes. 2 Tight morula and blastocysts/cleavage. 3 Blastocyst/total oocytes. 4 Blastocysts/cleavage. Table 2 Morula and blastocyst development rates (mean S:D:) over the total number of embryos produced among the two different oocyte and species sources (%) Embryos1 (n) Group A Group B Group C 95 291 497 Mor/tot embryos2 (%) 11.1 18b 22.3 12.3a 11.8 6.6b Bls/tot embryos3 (%) 88.9 18b 77.6 12.3a 88.1 6.6b

Columns with different superscripts (a, b) differ (P < 0:05). 1 Total number of embryos produced. 2 Morula rate over the total number of embryos produced. 3 Blastocyst rate over total number of embryos produced.

4. Discussion The in vitro embryo production efciency in buffalo species using oocytes recovered by means of transvaginal follicular aspiration was compared to in vitro embryo output obtained with oocytes originating from abattoir ovaries. We also compared the IVEP efciency of abattoir-derived oocytes in bovine and buffalo species. Ovum pick up was undertaken in a period of the year known to be unfavorable to the buffalo in terms of reproductive efciency [17,2931], and embryonic developmental parameters were compared to embryos derived from buffalo and bovine abattoir ovaries at the same time of the year. There was no specic reason for choosing that period of the year other than animals were available for carrying out the experiment. A higher proportion of Grade A oocytes were recovered from slaughterhouse ovaries. We speculate that this nding may be accounted for by the greater mechanical damage induced to granulosa cells during the OPU procedure. In fact the length of the needle, as well as of the line connected to the suction unit, may result in a greater loss of granulosa cells. In disagreement with this nding, within the buffalo species, despite a similar cleavage rate a higher embryo developmental rate was recorded in the OPU-derived oocytes. Furthermore, regarding the total embryo output between the two different source of oocytes, development of embryos derived from slaughterhouse ovaries was arrested at the morula stage at a signicantly higher rate compared to OPU-derived oocytes. This unexpected nding is not easily explained. The greater mechanical disruption which

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oocytes undergo during OPU might result in an improper evaluation of their quality, which is probably underestimated compared with abattoir-derived oocytes. However, the OPU technique results in resetting the follicular population and subsequently increasing the follicular wave frequency [11]. It follows that the occurrence of follicular atresia is highly reduced. In fact follicles are punctured before they become atretic. We also speculate that the cause of the different results may reside in an intrinsically higher sensitivity of buffalo oocytes to environmental stress. Oocytes recovered from slaughterhouse ovaries undergo a longer time interval between excision of ovaries from the peritoneal cavity and laboratory processing. On the contrary oocytes recovered by ovum pick up, which are immediately and properly processed on the spot and then moved to the laboratory for further processing, develop in greater proportions to the morula and blastocyst stages. Possibly, buffalo oocytes may be more affected or more quickly affected by cellular damage due to autolytic processes after residing for a prolonged period in excised ovaries. In oocytes derived from abattoir ovaries, cleavage rates were signicantly higher in cattle than in buffalo. Furthermore, signicantly higher morula and blastocyst rates as a proportion of the total number of COCs were found in bovine species, in accordance with previous reports [25]. However, for oocytes derived from abattoir ovaries, when late stage embryo development rates were calculated in relation to cleaved oocytes, results differed only for blastocyst output. What are the possible causes of lower embryo production efciency in buffalo compared with cattle? It cannot be ruled out that buffalo oocytes are more sensitive to environmental stress than bovine oocytes. The effect of stress seems to affect mainly postfertilization development, as demonstrated by the lower proportion of Tm that reached Bl stage at Day 7 when abattoir-derived oocytes were used. In addition to this we emphasize that no differences in cleavage rate were found within buffalo species between the two sources of oocytes. Furthermore, the entire in vitro system has been developed in the buffalo species by extrapolating information acquired in more studied species such as cattle. Progress has also been hampered by the shortage of experimental material such as ovaries from slaughtered animals, to use for research purposes in the countries in which the species is bred. The in vitro maturation step represents a landmark for further development. Although the incubation time is considered to be similar to the bovine species, there is some controversy regarding the required time for nuclear and cytoplasmic maturation in vitro [32,33]. In fact, our recent investigations have revealed a shorter time required for in vitro nuclear maturation in the buffalo species [34]. This aspect, together possibly with different embryo nutritional requirements in the early developmental steps and the lower quality of frozen semen [35], may account for the lower cleavage rates. Interestingly, despite a signicantly lower cleavage rate, buffalo OPU-derived oocytes developed to blastocysts at a similar rate to cattle abattoir-derived oocytes. Subsequently the highest blastocyst production, evaluated as a percentage of cleaved oocytes, was recorded in Group A. It is likely that OPU-derived buffalo oocytes are ``equipped'' with a more efcient molecular machinery, leading to the nal stages of preimplantation embryos in the buffalo species. On the contrary, previous reports in cattle have not shown a different developmental competence when the two sources of oocytes were compared [36]. This nding supports our hypothesis that buffalo oocytes are more sensitive to stress than cattle oocytes.

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As all procedures were carried out by the same technical personnel, these different results are believed to reect biological differences. Since differences among species decrease when results are calculated on cleaved oocytes, we believe that IVEP efciency in buffalo may be greatly improved by minimizing stress before IVM and identifying the causes of the lower cleavage rate. It is not clear if the lower cleavage rate is due to problems occurring during IVM or IVF. The improvement of these steps is therefore critical to optimize the efciency of advanced reproductive strategies in this species. Acknowledgements The authors are grateful to Prof. Robert H. Foote for critical reading of the manuscript. References
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