Animal Reproduction Science 64 (2000) 103112

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15C
J. Roca , S. Mart nez, J.M. Vzquez, X. Lucas, I. Parrilla, E.A. Mart nez
Department of Animal Pathology (Animal Reproduction), Veterinary Medicine, University of Murcia, Campus de Espinardo, 30.071 Murcia, Spain Received 14 March 2000; received in revised form 21 June 2000; accepted 21 June 2000

Abstract Articial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P < 0.05), irrespective of the extender used. There were interactions between extender and time of storage (P < 0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P < 0.05) and TTG the lowest (P > 0.05). In Experiment 2, a eld trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15 106 sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 C. Overall conception and farrowing rates were 77.1 0.7 and 70.4 0.7, respectively, and the mean litter size was 7.6 0.1. Fertility results were unaffected by the time of semen storage (P > 0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P < 0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving

Corresponding author. Tel.: +34-968364735; fax: +34-968367069. E-mail address: roca@um.es (J. Roca).

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viability and fertilizing capability of rabbit spermatozoa stored at 15 C. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Rabbit; Spermatozoa; Tris extender; Cool storage; Articial insemination

1. Introduction Articial insemination (AI) has been practised in the rabbit meat industry for over 15 years (Sinkovicks et al., 1983). In order to improve breeding management, AI is used routinely in many of the large rabbit farms in a number of European countries including Italy, France, Spain and Hungary. For practical application, rabbit AI is usually carried out with fresh diluted semen within 612 h of collection, resulting in conception rates as high as those obtained with natural mating. Despite the use of AI in the large rabbit farms of several European countries, rabbit AI has certainly not become a common practice in the rabbit meat producing areas of the world. A limiting factor for a more extensive commercial application is related to the semen preservation. The current practice of using freshly diluted semen is mostly limited to AI of does on the farm where the buck is located and within a few hours of semen collection. This short life places considerable constraints on rabbit AI since rapid delivery is essential. Since frozen semen is not currently suitable for routine AI in market rabbit production (for a review see Morrell, 1995 and Castellini, 1996), chilled and stored semen for 23 days may facilitate the semen transport and subsequent widespread use of articial insemination in rabbits. The aim of the present study was to evaluate the viability of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 C and to determine its conception and farrowing rates in a commercial breeding program in order to improve the appeal of using AI among meat producing rabbit industry.

2. Materials and methods 2.1. Animals All males and females were sexually mature hybrid rabbits. They were housed in at deck cages in rooms with controlled light (16:8 h L:N). Room temperature ranged from 16 to 28 C. Animals received a commercial diet (Purina ) according to their reproductive condition. Water was provided ad libitum. 2.2. General procedure 2.2.1. Semen collection, processing and assessment of spermatozoa viability Semen was collected four times a week with an articial vagina from male rabbits housed at a commercial AI center (CUNISANT, Murcia, Spain). After semen collection, any gel plug was removed. Only ejaculates with good wave motion (3 on a 05 scale) were used.

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The concentration of spermatozoa was 300 106 cells ml1 and the proportion of motile cells was 75%. The assessment of spermatozoa viability included the percentage of total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (normal apical ridge, NAR). To evaluate the TSM percentage and FPM score, three samples of the diluted spermatozoa after 10 min incubation at 37 C were placed under a coverslip in the center of a pre-warmed (39 C) slide and transferred to a heated microscope stage set at 39 C and subjectively assessed by phase contrast microscopy (100 and 200 magnications). The TSM was estimated in ranges of 5% and the proportion of spermatozoa with FPM was determined using an arbitrary scale of 05 (0, 1, 2, 3, 4 or 5 = 010, 1025, 2550, 5070, 7090, or 90100%, respectively, of the motile spermatozoa showing progressive movement). The percentage of spermatozoa with PMI was evaluated using a uorescence microscope (magnication 400) using a standard uorescein lter set (Nikkon, Japan) after staining of spermatozoa with 6-carboxyuorescein diacetate (Sigma Chemical, St. Louis, MO, USA) as described previously by Harrison and Vickers (1990). Percentage of spermatozoa with NAR was assessed by viewing wet mounts of diluted spermatozoa xed in buffered 2% glutaraldehyde solution (Pursel and Johnson, 1974). NAR was examined by phase-contrast microscopy using a 100 oil immersion objective. For PMI and NAR evaluation, a total of 200 spermatozoa were counted. 2.2.2. Synchronization of oestrous, induction of ovulation, insemination and pregnancy diagnosis Female rabbits housed in a commercial farm were induced to oestrous synchronously with subcutaneous application of PMSG (20 UI, Folligon , Intervet, Holland) 5256 h before AI. To induce ovulation, does were injected i.m. with 0.8 g of gonadotropin-releasing hormone analogue (Buserelin, Suprefact , Hoechst-Roussel, Germany) at the time of insemination. Inseminations were performed at one time in a xed day of the week with a dose of 0.5 ml of diluted semen containing 15 106 spermatozoa. The insemination procedure was similar to that described by Boussin (1989). AI was performed in nulliparous, primiparous and multiparous does. Nulliparous does were inseminated at 1722 weeks of age, primiparous and multiparous does were inseminated 11 days after parturition. Pregnancy diagnosis (conception rate) was determined by abdominal palpation on day 14 after AI. Non-pregnant does were inseminated again 21 days after the rst non-fertile insemination (re-breed does). The farrowing rates and litter size were recorded at the time of farrowing. 2.3. Experimental design 2.3.1. Experiment 1 The objective of the rst experiment was to evaluate the viability of rabbit spermatozoa diluted in Tris-based extenders and stored at 15 C for 96 h. Semen was collected from 12 male rabbits and ejaculates with adequate motility and sperm concentration were pooled and divided into four aliquots. Each one of the four aliquots was extended to 3040 106 spermatozoa ml1 (1:10, semen/extender) in one of the following extenders: Tris-citric-glucose (TCG), Tris-citric-fructose (TCF), TES-Tris-glucose

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Table 1 Chemical composition of Tris-buffer extenders used to dilute and preserve rabbit semen at 15 C Components (mM) Extendera TCG Trisb TESc Citric acid Glucose Fructose Kanamycin (mg/l) pH Osmotic pressure (mOsm/l)
a b

TCF 313.79 103.07 33.3 80 6.9 333

TTG 56.15 238.09 33.3 80 6.9 348

TTF 56.15 238.09 33.3 80 6.9 344

313.79 103.07 33.3 80 6.9 336

TCG: Tris-citric-glucose, TCF: Tris-citric-fructose, TTG: TES-Tris-glucose and TTF: TES-Tris-fructose. Tris: Tris(hydroxymethyl)aminomethane. c TES: N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

(TTG) and TES-Tris-fructose (TTF) (Table 1). The extended semen samples were cooled slowly and held at 15 C for 96 h. Assessment of spermatozoa viability was determined 0, 24, 48, 72 and 96 h after the beginning of cooling. This experimental procedure was repeated six times. 2.3.2. Experiment 2 The objective of the second experiment was to determine the fertility rates of spermatozoa diluted in a Tris-based extender and stored at 15 C between 0 and 48 h. Results of the Experiment 1 led us to choose the TCG extender to use in this fertility trial because it maintained adequate sperm viability over 48 h, and was commercially affordable. Semen from 30 rabbits was used in this experiment. After collection and evaluation of semen quality, pooled gel-free ejaculates were diluted to 30 106 spermatozoa ml1 in TCG extender. AI was performed once using either fresh diluted semen (n: 1275) and diluted semen stored at 15 C for 24 h (n: 1503) or 48 h (n: 935). Inseminations were performed throughout the 12 months and fertility was measured by conception rate (percentage of pregnant rabbits after palpation), farrowing rate and litter size. 2.4. Statistical analysis Calculations were made using the SPSS 9.0/PC statistics package (SPSS Inc., Chicago, IL, US). Data from Experiment 1, after arcsin transformation of the percentages, were examined by ANOVA using a repeated-measures general linear model where storage time was the repeated measure. Means S.E.M. for sperm parameters were compared using Duncans multiple range test. In Experiment 2, data from both conception and farrowing rates (the data were modeled according to the binomial model of parameters, as described by Fisz, 1980) and litter size were analyzed by two-way analysis of variance using a general linear model. When ANOVA revealed a signicant effect, values were compared using Tukey-test. Differences were considered to be statistically different at P < 0.05.

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3. Results 3.1. Experiment 1 The effects of storage time at 15 C on the viability of spermatozoa diluted in the four Tris-buffer extenders are presented in Fig. 1. Regarding all sperm parameters, the viability of spermatozoa declined with time of storage (P < 0.05), irrespective of the extender used. There were interactions between extender and time of storage (P < 0.05) on all viability parameters evaluated. After 96 h of storage, TTG provided signicantly (P < 0.05) lower sperm viability than the other Tris-buffer extenders used. When comparing extenders within the same time of storage, no signicant differences (P > 0.05) were found between spermatozoa examined directly after dilution (0 h) with regard to all sperm viability parameters. However, TSM and PMI at 24 h and TSM, FPM and PMI at 48 h were lower (P < 0.05) for spermatozoa in TTG than for spermatozoa diluted in the other Tris-buffer extenders. By 72 h, TSM, FPM and PMI were higher (P < 0.05) for spermatozoa diluted in TCG than in TTG. Spermatozoa examined at 96 h, showed the highest (P < 0.05) TSM, FPM and PMI when diluted in TCG. Extender had no effect on the percentage of NAR (P > 0.05).

Fig. 1. Mean (S.E.M.) percentage of total sperm motility (a), forward progressive motility (b) and percentages of sperm with plasma membrane integrity (c) and normal acrosome (d) of rabbit spermatozoa at different times of preservation at 15 C after dilution in Tris-citric-glucose (), Tris-citric-fructose (), TES-Tris-glucose () and TES-Tris-fructose () extenders. Means marked with asterisk are signicantly different ( P < 0.05; P < 0.01).

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3.2. Experiment 2 Inseminations with semen diluted in TCG extender were made on a total of 3713 does. The overall conception and farrowing rates were 77.1 0.7 and 70.4 0.7, respectively, and the mean litter size was 7.6 0.1. Table 2 shows the fertility results according to the time of semen storage. Since the physiological condition of does had a signicant effect on the fertility results (P < 0.001), the does inseminated in each semen storage time were grouped according to their physiological condition. There was no interaction between time of semen storage and physiological condition of does (P < 0.05) in any of fertility parameters evaluated. There were no signicant differences (P > 0.05) in either the conception rate or the farrowing rate or the litter size between the different semen storage times. Regardless of semen storage time, lactating and nulliparous does showed the highest conception and farrowing rates (P < 0.01) and primiparous the lowest (P < 0.05).

4. Discussion The effective use of chilled semen for AI depends upon the ability of the extender to provide a suitable environment for spermatozoa during storage. One basic component of semen extender in the biological buffers to minimize pH uctuation due to metabolic by-products of both sperm cells and contaminant bacteria (Watson, 1990). Different buffers have been tested to evaluate their capability to keep chilled rabbit semen over time (for review see Castellini, 1996). This author concluded that inorganic buffers have a limited buffering capability, whereas organic buffers, as Tris, are more appropiate for the storage of rabbit semen at low temperatures. Tris-buffer extenders have been found to be useful in the short-term storage (6 h) of chilled rabbit semen (Maertens and Luzi, 1995). However, experiments to evaluate their use in prolonging sperm viability and fertility throughout time are very limited. El-Gaafary (1994) using a Tris-yolk extender found that spermatozoa cooled and stored at 5 C for 24 h had a mean motility of 45%, and after 48 h fell to 25%. This sharp decline in sperm viability throughout the preservation time might be due to chilling temperature. The temperature at which perturbation of the rabbit sperm cell occurs has not been established, however studies comparing various temperatures conclude that 15 C is more appropiate than 5 C to store chilled rabbit semen (for review see Castellini, 1996). Results of our in vitro study demonstrated that 15 C can be an adequate temperature to store chilled rabbit semen when Tris-buffer extenders are used. So, in vitro results of Experiment 1 shown as the TSM, PMI and NAR were still over 75% and a FPM higher than 3 (05 scale) at 48 h of storage when rabbit spermatozoa were diluted in TCF, TCG and TTF. Inclusion of TES into Tris-buffer extenders for the dilution of mammalian spermatozoa has been suggested since it enhances the buffer capacity of Tris (Graham et al., 1972). The combination TES-Tris has been used with success as the basis of diluents for small ruminant semen (Maxwell and Salamon, 1993). Its potential effectiveness in improving the sperm viability and fertility of preserved rabbit semen has not previously been evaluated. In our in vitro experiment, TES-Tris extenders were no better than Tris alone extenders. Thus, since TES is expensive, its inclusion into Tris-buffer extenders to dilute rabbit semen was not justied.

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Glucose and fructose were the main sugars added to Tris-buffer extenders (Watson, 1990). Costantini (1989) found that rabbit sperm viability was higher when glucose was used. In Experiment 1, no differences were found on sperm viability when rabbit semen was diluted in TCG and TCF extenders, however semen diluted in TTG extender showed the lower sperm viability after 96 h of storage at 15 C. The reason of this decline cannot be attributed exclusively to the glucose since properties of added sugars are largely dependent on interactions with the other constituents in the extender and it might be that Tris-TES performs better in vitro when combined with fructose rather than glucose. An important negative factor responsible for the reduction in sperm longevity are the metabolic by-products of uncontrolled bacterial growth since rabbit ejaculates tend to be contaminated with bacteria during the semen collection process. Bacterial growth can be signicantly limited by added antibiotics to extender. Many antibiotics have been recommended and used in semen extenders, however, their effectiveness in the preservation of rabbit semen has not been evaluated. Kanamycin is a broad-spectrum antibiotic and works well as a preservative in boar semen stored at 15 C (Althouse, 1997). Therefore, it was included in the Tris-buffer extenders used in the present study. Although kanamycin was effective in our experiments since no bacterial growth was seen during sperm evaluation process, it might be expensive for the routine eld use with rabbit semen. Further studies to select appropriate antibiotics for chilled preservation of rabbit semen are required. To perform a fertility trial under controlled eld conditions, inseminations were made in a commercial farm where the rabbit does were unselected and the semen used provided from routinely collected ejaculates from male rabbits standing at the commercial AI centre. Since the accurate assessment of the fertility of a semen sample requires large numbers of inseminations and the commercial farm where the Experiment 2 took place is not very large, just one of the extenders in vitro evaluated on Experiment 1 was used. So, TCG was selected according to both their capability to preserve sperm viability over time and to its cost. Previous studies to evaluate fertility of rabbit stored semen have been carried out under experimental conditions (El-Gaafary, 1994; Maertens and Luzi, 1995). Whereas Maertens and Luzi (1995) showed that conception rate did not decrease when semen was stored at 20 C during 6 h after dilution in commercial Tris-based extender, El-Gaafary (1994) showed a signicant fall on the farrowing rates when semen was stored at 5 C for 24 and 48 h after dilution in a Tris-yolk extender. In contrast, our eld study showed that Tris-citric-glucose extender is effective in retaining the fertilizing capacity of rabbit spermatozoa stored at 15 C up to 48 h. The difference between El-Gaafarys study and the one reported here is that we did not cool semen below 15 C. The temperature of semen storage is an important factor to retain the fertilizing capacity of rabbit spermatozoa (Lpez et al., 1996) and 15 C appears to be better than 5 C when Tris-buffer extenders are used. An important factor inuencing conception rates is the number of sperm inseminated. The lack of signicant differences between fresh and stored semen on conception rates for Experiment 2 may be explained by the number of sperm used per doses (15 106 cells). Several authors have conducted studies to determinate the minimum optimal number of total sperm inseminated (Farrell et al., 1993; Viudes de Castro and Vicente, 1997, Alvario et al., 1998). Farrell et al. (1993) concluded that many conditions inuence the minimal number of sperms required for maximal conception rates in rabbits, among these is included

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the extender and time of semen storage. Whereas Viudes de Castro and Vicente (1997) suggested that an inseminate dosis with 4 million is enough to allow adequate conception rates when fresh semen was used, Alvario et al. (1998) suggest that the lowest advisable limit should be over 20 million with 24 h preserved semen. Our results with stored semen suggest that the number of sperms could be slightly less (15 million). This was probably due to the good sperm quality of stored semen used in our study. Semen doses used in eld trial of Experiment 2 always had a percentage of motile sperm up to 75%. Both conception and farrowing rates in rabbit farms are inuenced by the physiological conditions of does (for a review see Castellini, 1996). In the present study, four categories of does with different reproductive responses have been dened: nulliparous, primiparous, lactating and re-breed does. Regardless of time of semen storage in Experiment 2, nulliparous and lactating does showed the highest conception and farrowing rates and primiparous gave the lowest. The poor reproductive response of primiparous does is well-known (Castellini, 1996). This is related together with hormonal status and energy decit which have a detrimental effect on foetal survival (Fortun and Lebas, 1994). However, the lack of signicant differences between nulliparous and lactating does on conception rates and farrowing rates is in contrast to the results previously reported (Rebollar et al., 1992). They reported that fertility of nulliparous does is higher than lactating does. The disagreement may be related to the parturition-insemination interval. Whereas Rebollar et al. (1992) inseminated does at day 34 post-partum, we performed the inseminations at day 11. Theau-Clment and Roustan (1992) reported that the antagonism between lactation and ovulation frequency is closely related to the stage of lactation and it is more pronounced during the rst week after parturition. They concluded that farrowing rates were highest when does were inseminated 1012 days after parturition. It can be concluded that Tris-buffer extenders are effective for dilution and storage of rabbit semen at 15 C, and that Tris-citric-glucose extender retain the fertilizing capability of rabbit spermatozoa through 48 h when they are stored at 15 C.

Acknowledgements The authors are indebted to Gloria Riera and Ramn Montao for their excellent technical assistance. The staff at Cunisant in Murcia are also acknowledged for their excellent cooperation thoughout the study. This study was funded in part by Purina Espaa S.A. Silvia Mart nez was supported by a grant from the Spanish Ministry of Education and Culture.

References
Althouse, G.C., 1997. Comparison of currently used semen extenders in the swine industry. Compendium 19, 777782. Alvario, J.M.R., Lopez, F.J., Del Arco, J.A., Bueno, A., Torres, R., 1998. Effect of semen concentration on rabbit articial insemination with fresh or 24 h stored semen. In: Proceedings of 6th World Rabbit Congress, Toulouse, France, Vol. 1, pp. 3335. Boussin, D., 1989. Reproduction et insemination articielle en cuniculture. Association Francaise de Cuniculture, Lempdes, Paris.

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J. Roca et al. / Animal Reproduction Science 64 (2000) 103112

Castellini, C., 1996. Recent advances in rabbit articial insemination. In: Proceeding of 6th World Rabbit Congress, Toulouse, France, Vol. 2, pp. 1326. Costantini, F., 1989. Fecondazione articiale nel coniglio, sistemi di conservazione dello sperma. Riv. Coniglicolt. 26, 1418. El-Gaafary, M.N., 1994. Quality and fertility of cooled rabbit semen supplemented with cyclic-AMP stimulators. Anim. Reprod. Sci. 34, 307313. Farrell, P.B., Foote, R.H., Simkin, M.E., Clegg, E.D., Wall, R.J., 1993. Relationship of semen quality, number of sperm inseminated and fertility in rabbits. J. Androl. 14, 464471. Fisz, M., 1980. Some probability distributions. In: De Krieger, R.E. (Ed.), Probability Theory and Mathematical Statistics. Wiley, Malabar FL, USA, pp. 129174. Fortun, L., Lebas, F., 1994. Inuence of the number of suckling rabbit young and the feed level on foetal survival and growth in rabbit does. Ann. Zool. 43, 163171. Graham, E.F., Crabo, B.G., Brown, K.I., 1972. Effects of some zwitterion buffers on the freezing and storage of spermatozoa. I. Bull. J. Dairy Sci. 55, 372378. Harrison, R.A.P., Vickers, E., 1990. Use of uorescent probes to assess membrane integrity in mammaliam spermatozoa. J. Reprod. Fertil. 88, 343352. Lpez, J., Alvario, J.M.R., Del Arco, J.A., Delgado, F., Ramiro, J.L., 1996. Effect of cooling temperature on 24 h stored semen for articial insemination of rabbit. In: Proceedings of 6th World Rabbit Congress, Tolouse, France, Vol. 2, pp. 7982. Maertens, L., Luzi, F., 1995. Effect of diluent and storage time of rabbit semen on the fertility of does reared under two different lighting schedules. World Rabbit Sci. 3, 2734. Maxwell, W.M.C., Salamon, S., 1993. Liquid storage of ram semen. Reprod. Fertil. Dev. 5, 613638. Morrell, J.M., 1995. Articial insemination in rabbits. Br. Vet. J. 151, 477488. Pursel, V.G., Johnson, L.A., 1974. Glutaraldehyde xations of boar spermatozoa for acrosome evaluation. Theriogenology 1, 638641. Rebollar, P.G., Ubilla, E., Rodr guez, J.M., 1992. Inuence of the parturition-insemination interval on the conception rate in rabbits articially inseminated with fresh semen. J. Appl. Rabbit Res. 15, 407411. Sinkovicks, G., Medgyes, I., Paljak, J., 1983. Some results of articial insemination in rabbits. J. Appl. Rabbit Res. 6, 4348. Theau-Clment, M., Roustan, A., 1992. A study on relationships between receptivility and lactation in the doe, and their inuence on reproductive performances. J. Appl. Rabbit Res. 15, 412421. Viudes de Castro, M.P., Vicente, J.S., 1997. Effect of sperm count on the fertility and prolicity rates of meat rabbits. Anim. Reprod. Sci. 46, 313319. Watson, P.F., 1990. Articial insemination and the preservation of semen. In: Lamming G.E. (Ed.), Marshalls Physiology of Reproduction. Vol. 2. Reproduction in the Male. Churchill Livingstone Editorial, Edinburgh, pp. 747869.