Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition

ELSEVIER

POST-THAW SURVIVAL OF RAM SPERMATOZOA AND FERTILITY AFTER INSEMINATION AS AFFECTED BY PREFREEZING SPERM CONCENTRATION AND EXTENDER COMPOSITION A.G. D'Alessandro, la G. Martemucci! ,2 M.A. Colorma 3 and A. Bellitti 3 IDepartment PRO GE.SA., University of Bari, Italy 2Research Center on Small Ruminants, CNR, Bari, Italy 3Department of Animal, Vegetal and Environmental Sciences, University of Molise, Italy Received for publication: A p r i l 19, 2000 Accepted: October 18, 2000 ABSTRACT A study was conducted to investigate the effects ofprefreezing sperm concentration using two extenders on post-thaw survival and acrosomal status of ram spermatozoa (Experiment 1) and fertility after intrauterine insemination with differing doses of semen (Experiment 2). In autumn (Northern hemisphere), semen was collected by artificial vagina from 8 adult Leccese rams and ejaculates of good quality semen were pooled. Two extender systems for cryopreservation were considered, one based on milk-lactose egg yolk (Milk-LY) and the other based on tris-fructose egg yolk (Tris-FY). Experiment 1 (2 x 6 factorial scheme) examined the in vitro characteristics of spermatozoa in relation to the Milk-LY and Tris-FY extenders and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x l06 spermatozoa/mL). Experiment 2 (2 x 4 factorial) evaluated the influence of the Milk-LY vs Tris-FY extenders and four doses (20, 40, 80 and 160 x 106 spermatozoa/0.25 mL) corresponding to prefreezing spermatozoa concentrations of 100, 200, 400 and 800 x 106 spermatozoa/mL, on fertility of ewes inseminated in uterus by laparoscope. Prefreezing sperm concentration influenced (P < 0.01) freezability of spermatozoa and affected negatively all the in vitro parameters at 800 x 106 spermatozoa/mL. Overall, MilkLY tended to ensure higher viability and acrosomal integrity of spermatozoa after thawing at the intermediate sperm densities (range 100 to 500 x 106 spermatozoa/mL). At 500 x 106 spermatozoa/mL concentration corresponded the best condition for survival of spermatozoa (71.2%), acrosome integrity (71.5%) and acrosomal loss (6.0%). At the lowest sperm concentration (50 x 106 spermatozoa/mL), Tris-FY resulted in a higher survival rate than MilkLY (61.3%, P < 0.05) and lower acrosomal loss (9.7%, P < 0.05). Milk-LY supported spermatozoa motility better than Tris-FY after incubation at sperm concentration between 50 and 400 x 106 spermatozoa/mL (0.05 > P < 0.01). Semen doses of 20 to 40 x 106 spermatozoa/ewe provided satisfactory fertility rates (64 to 81%). The increase of inseminate doses to 160 x 106 spermatozoa/ewe failed to improve fertility, actually tending to decrease lambing rates. © 2001 by Elsevier Science Inc

Key words' extender, sperm concentration, viability, fertility, ram semen Acknowledgement This work was supported by National Research Council of Italy from Research Center on Small Ruminants, Bari. The authors thank L. Bongermino for statistical processing analysis and L. Basso and P. Cataldo for technical assistance in conducting the experiments. "Correspondence and reprint requests. Thenogenology 55:1159-1170, 2001 © 2001 Elsevier Science Inc.

O093-691X/O1/$-see front matter PII: S0093-691X(01)00474-5

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INTRODUCTION The development of cryopreservatlon technology for ram spermatozoa made considerable progress toward practical and economic application of artificml insemination (AI) under field conditions. Many factors can affect the maintenance of spermatozoa function during freezing and thawing, such as freezing method, equilibration periods and coohng rate (3, 6, 22). Moreover, composition of the extender and prefreezmg dilution rate may be pivotal relative to freezabflity of spermatozoa and their fertd~zmg ability. The different techniques used in the deposition of semen m the reproductxve tract of the females required considerable numbers of spermatozoa to provide sattsfactory results, In cerwcal insemination, a semen dose characterized by low volume but high spermatozoa concentration is required (1, 29), while for the deposmon of semen directly in utero by laparoscope or transcervical Guelph-System, lower doses are required (12, 13, 20, 39) Many kinds of diluents, originally used in most cases for freezing bull semen, and different combinations among the components are used for preservation of ram spermatozoa (23, 30). However, the freezing procedure reduces motility and viability of spermatozoa requiring many inseminate doses to be discarded after thawing (5) The aim of this study was to investigate the effects of prefreezing sperm concentration and the ability of two extenders based on bovine skim-milk or tris (tris(hydroxymethyl)amino methane) to support post-thaw survival and acrosomal integrity in ram spermatozoa. Laparoscopic insemination was also performed to assess fertility of the frozen semen with respect to both the extenders and inseminate doses corresponding to different prefreezing sperm concentrations. MATERIALS AND METHODS Experimental Design Two experiments were conducted m Southern Italy (41 ° N) with the aims of evaluating in vxtro post-thaw survival and acrosomal integrity (Experiment 1) and fertility after laparoscopic insemmauon (Experiment 2) of semen frozen according to two freezing systems based on MilkLY (6) and Tris-FY (18) This method, originally performed on buck semen, was modified slightly ehminating the wash step needed to remove seminal plasma from spermatozoa (20). Semen Collection and Processing During the breeding season (autumn), semen was collected from eight adult Leccese dairy breed of rams by artificial vagina. A single semen ejaculate usually was collected from each male twice a week Only ejaculates with a mimmum concentratLon of 3 x 109 spermatozoaJmL and 70% progressively motile cells were pooled across rams Two replicates of pooled semen were made for each sperm concentration within the two extenders. Semen extention using Milk-LY was performed m two steps (6). Briefly, the first dilution was carried out at 30 ° C with a solution prepared by mixing 20% (v/v) egg yolk and 80% (v/v) lactose soluuon 230 mM (pH = 6.6 to 67, 450 mOsm). The second one at 4 ° C with a glycerolate diluent prepared from a base solution consisted of 100 mL double-distilled sterile water with 11.11 g powder skim-milk (0.8 g fat milk per liter; Regilait, St. Martin Belle Roche, France), 0 33 g sulphanilamide plus pemcillin (0.06 g) and streptomycin sulphate (0.1 g) Thus, the second diluent was prepared from base solution adding other 4.0 g skim-milk and 1 mL

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tnsodium-citrate x 5.5 H20 (800 mM) and substituting 10 mL of this solution with 10% (v/v) glycerol. Semen dilution was performed so that final concentration of glycerol was 4%, regardless of sperm concentration achieved before freezing. The freezing system based on Tris-FY was performed according to Kupfetschmied and Muther (18) method. Semen was extended in two steps, at 30 ° C and 4 ° C and with diluent A and B, respectively. Diluents A and B were prepared from a base solution 300 mM tris, 100 mM citric acid anhydrous, 82.6 mM fructose, 1435 U/mk penicillin and 1132 U/mL streptomycin sulphate (pH = 7.2 to 7.4; 300 to 325 mOsm). Then, diluent A consisted of 67.2% (v/v) base solution, 12.8% (v/v) double-distilled sterile water and 20% (v/v) egg yolk while diluent B was obtained substituting 12.8% (v/v) water of diluent A w~th 12.8 % (v/v) glycerol. The diluents A and B were added respectively in 60% and 40% of the final volume required to obtain the different prefreezing sperm concentrations. Final glycerol concentration ranged from 3.5% to 5.0% in relation to the sperm concentratzons Semen extended in either milk-based diluent or Tris-based diluent to final sperm concentration of 50, 100, 200, 400, 500, or 800 x 106 spermatozoa/mL were loaded into 0.25 mL straws (I.M.V, L'Aigle, France) Straws were cooled in liquid nitrogen vapour and then plunged in liquid nitrogen (-196 ° C) for storage until required for analysis of semen. Experiment 1 Experiment 1 was a (2 x 6) factorial design where the factors were two extenders (Milk-LY and Tris-FY) and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x 106 spermatozoa/mL), corresponding to frozen semen doses containing respectively I0, 20, 40, 80, 100 or 160 x 106 spermatozoa/0 25 mL straw. After thawing (37 °C, 30 sec), assessment of survival and acrosomal integrity of spermatozoa was performed by simultaneous staining with Hoechst 33258 (Sigma; Milan, Italy) to identify dead or damaged spermatozoa, and fluorescein isothiocyanate combined with Pisum Sativum Agglutinin (FITC-PSA; Sigma), according to the method described by Cross et al. (11), with some modifications. Briefly, semen was diluted with PBS (phosfate buffer saline) to a concentration of 50 x 106 spermatozoa/mL, exposed to 50 ~tL H33258 (10 pg/mL in PBS) for 15 min at room temperature and in the dark, and finally washed with PBS twice by centrifugation at 2800 rpm (10 minutes) and resuspension. The sperm pellet was smeared on a glass microscope slide and fixed in 95% ethanol for 30 min at 4 ° C. Slides were washed in PBS and exposed to PSA labeled with FITC (10 mg/mL in PBS; Sigma) for 15 min in the dark and humidified air conditions at room temperature. The slides were washed in PBS and mounted with glycerol soluted in PBS (1'1, v/v). Within 2 h, 150 sperm cells were counted in duplicates for each sample under a fluorescence microscope (Dialux 20; Leitz, Germany) with xl000 (oil lens magnification) using LP 430 (340 to 380 nm) and LP 520 (450 to 490 nm) filters for H33258 and FITC-PSA, respectively. For acrosomal status, spermatozoa were scored as normal when the head was bright and uniformly fluorescing, damaged when the head was not completely fluorescing, without acrosome when the head was not fluorescing and spermatozoa with widespread fluorescence when the whole cell was fluorescing. The percentage of motile spermatozoa was assessed on a warm stage (37 ° C) after thawing (0 h) and after incubation at 37 ° C for I and 3 h. The percentage of motile spermatozoa was determined from estimates of relative proportions of motile and non-motile cells using a Makler counting chamber. Each speciment was observed on forty Makler cells under a binocular microscope (x 200) equiped with a heating (37 ° C) stage, with the support of a Polaroid

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Freezmg-Frame blocking image All samples were examined by two observers, with the average results used for subsequent analysis, and for two rephcations. Expertment 2 In Experiment 2 the insemination was conducted during the breeding season (autumn) according to a (2 x 4) factorial design corresponding to the two extenders of Experiment 1 (MilkLY and Tris-FY) and four prefreezing sperm concentrations (100, 200, 400 and 800 x 106 spermatozoa/mL), corresponding to the experimental inseminate doses of 20, 40, 80 and 160 x 106 spermatozoa/0.25 mE straw. Two hundred and forty-one mature Leccese dairy ewes from commercial flocks were randomly allocated to eight groups according to the factorial design. Estrus was synchronized with intravaginal pessaries containing fluorogestone-acetate (FGA; Chronogest; Intervet, Milan, Italy) left in sttu for 9 days. On the seventh day after pessary insertion, the ewes received 50 gg prostaglandin F2ct, (PGF2ct; Cloprostenol, ICI, Schering Plough, Milan, Italy) and 400 UI PMSG (Intervet, Milan, Italy) by im injections. The ewes were inseminated in uterus by laparoscopy 43 h after sponge removal (20). Of the inseminate volume (0.25 mL), one half was deposited into the lumen of each horn, approximately midway between the uterine body and the utero-tubal junction At lambing, fertility was recorded. Statistical Analysis The data were examined by the Bartlett test for homogenmty of variance and, where not normally distributed, were transformed to log or arcsine function before statistical analysis. All analyses were performed using the GLM procedure of SAS (33). Data were subjected to analyses of variance for a split-plot design with the extenders and prefreezing sperm concentrations as the main plots. For the analysis of percentage of motile spermatozoa at thawing and after 1 and 3 h incubation, incubation times were included in the statistical model as subplots. Data regarding post-thaw sperm motility are presented as means over the three observation times plus or minus root-MSE error. Data regarding percentages of live spermatozoa and spermatozoa with integral or damage acrosome are presented as means from readings conducted at one observation time. Least squares means were compared by the predicted difference (pdiff) option of GLM (33). Differences of fertility, related to the inseminate doses of semen, were analyzed by the chi-square test (33). RESULTS Experiment 1- In Vitro Characteristics of Semen Percentage of live spermatozoa after thawing assessed by exclusxon of Hoechst 33258 was influenced by prefreezing sperm dilution (P < 0.01) and the interaction sperm concentration by extender was also s,gmficant (P < 0.0l) Within the freezing system based on the use of Milk-LY extender, the prefreezing dilution rate to 500 x 106 spermatozoa/mL affected the highest proportion of live spermatozoa (71.2%, P < 0.01) (Table 1). The increase of sperm concentration to 800 x 106 spermatozoa/mL influenced negatively the survival of spermatozoa extended in Milk-LY (49.2%, P < 0.01) as well as in Tris-FY (47.9%, P < 0.01).

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The interaction spermatozoa concentration by extender indicated a higher survival rate of spermatozoa with Tns-FY extender tbr the concentration of 50 x 106 spermatozoa/mL (P < 0.05) and higher values with Milk-LY for 100 x 106 spermatozoa/mL (P < 0.01) and 500 x 106 spermatozoa/mL (P < 0.05) (Table 1) The proportion of spermatozoa with intact acrosome was not influenced by either pre-freezmg sperm concentration or extender The highest proportion of spermatozoa with intact acrosomes was observed m samples frozen in Mdk-LY at the concentration of 500 x 106 spermatozoa/mL (71 5%) being significantly different compared to 800 x 106 spermatozoa/mL (35 5%, P < 0.01 ) (Table 1). Considering the acrosomal damage to spermatozoa ('fable 1), a sigmficant effect of sperm concentration (P < 0 01) and its interaction with extender (P < 0 05) on loss of acrosome was found Using MIIk-LY extender, at lowest sperm dilution (800 x 106 spermatozoa/mL) there was the highest propomon of spermatozoa without acrosome (23 l%, P < 0.01) Semen frozen at 500 or 200 x 106 spermatozoa/mL showed significantly (P < 0 0l) lower values compared to 50 x 106 spermatozoa/mL. Also for Tris-FY, the highest concentration of spermatozoa (800 x 106 spermatozoa/mL) reflected the highest incidence of acrosomal loss (15 6o, P < 0.01) compared to 50 and 400 x 106 spermatozoa/mL. The influence of the extender on acrosomal loss was marked m spermatozoa frozen at the extreme concentrations (50 and 800 x 106 spermatozoa/mE) for which there were the lowest percentages of spermatozoa wtthout acrosome m Tris-FY (9.7 and 15 6%) compared to the Milk-LY extender (15.7 and 23 I%,P < 0 0 1 ) The mean percentage of motile spermatozoa after thawing and incubation at 37 °C was affected (P < 0 01) by both prefreezing sperm dilutton and extender and their interactton was also slgmficant (P < 0 05) (Table 2). At thawing, Mdk-LY resulted m the highest proportion of motde spermatozoa when spermatozoa were frozen at the concentration of 100 x 106 spermatozoa/mL (83.3%) and, although durmg incubation thin percentage decreased to 69.0% (P < 0 05), it remained the highest value (mean 0 to 3 h = 77.4%) The increase of sperm concentration to 800 x 106 spermatozoa/mL resulted m the lowest post-thaw motdity (37.9%, P < 0 01 ) and a more rapid dechne in the motdtty of spermatozoa during 3 h mcubatton (12 9%, P < 0.01). Slmdarly, the spermatozoa concentration of 500 x 106 spermatozoa/mL motile spermatozoa declined to 44.0% after the incubation time (P < 0.01). In addition to semen frozen in Tns-FY extender, the highest prefreezing cell concentration had a detrimental effect on sperm motdlty both at thawing (51.1%, 0.05 > P < 0.01) and after 3 h incubation (12 4%, P < 0.01). At this time, the highest percentage of motile spermatozoa was seen in the semen frozen at 50 x l06 spermatozoa/mE (52 7%) with significant differences (P < 0 01) compared to 100, 400 and 800 x 106 spermatozoa/mL. The interaction between extender and prefreezing sperm coneentrauon indicated that Mdk-LY improved sperm motdlty in semen frozen at lower concentrations compared to Tris-FY, respectively in correspondance to 100 x 106 spermatozoa/mL at thawing (P < 0.01) and to 100, 200, 400 (P < 0.01) and 50 x 106 spermatozoa/mL (P < 0 05) after 3 h incubation Tris-FY gave higher sperm motility at thawing for the highest prefreezing sperm concentratton (800 x 106 spermatozoa/mL) than Milk-LY (P < 0.05).

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No differences were found between the two extenders for the sperm concentrations of 500 and 800 x 106 spermatozoa/mL after 3 h incubation. Experiment 2: Fertihty after laparoscopic insemination The results of this study, regardmg reproductive performance of ewes after intrauterine insemination, are reported in Table 3 Fertdlty was not affected by either inseminate dose, corresponding to different prefreezmg concentrations of spermatozoa, or extender. Data pooled across the extenders indicated a tendency of spermatozoa in Tris-FY to g~ve higher fertility compared to those in Milk-LY extender (70.2 vs 61.7%, P > 0.05). Fertility tended to be highest (P > 0 05) at the lowest doses of spermatozoa (20 and 40 x 106 spermatozoa/ewe) frozen in TrisFY (71.1 to 81%). Fertility decreased at the maxtmum of 160 x 106 spermatozoa (57.1%) in Milk-LY (54.8%, P > 0.05) Table 3.

Extender Milk-LY

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Lambing rates of ewes after intrauterine insemination wtth different doses of semen corresponding to different pre-freezing spermatozoa concentrations tn milk-lactose egg yolk (Milk-LY) or tris-fructose egg yolk (Tris-FY) based extender. Pre-freeze spermatozoa concentration (x 106/mL)

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DISCUSSION Cryopreservation of semen induces a series of structural and biochemical modifications in spermatozoa thereby reducing thetr membrane integrity (4, 15, 25, 34, 35, 40), motility (17, 25) and fertilizing ability (24, 37, 38). Various methods for processing and freezing of spermatozoa were developed to reduce cryogenic injuries to spermatozoa (30). Much of the physical damage to spermatozoa affects plasma and acrosomal membranes (26) The physical damage to spermatozoa decreases their functionahty because of loss of cellular components including acrosin (4, 10). In thxs study Milk-LY extender provided more effective cryopreservation of both plasma and acrosomal membranes in semen diluted to final concentrations from 100 to 500 x 106

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spermatozoa/mL before freezing compared to the Tris-FY extender. Moreover, these findings support the positive relationship between spermatozoa with intact membrane and acrosome integrity found in our previous study in bucks (21) and in rams by Valcarcel et al. (35). For both the extenders used in this study, spermatozoal acrosome integrity was always lower than the proportion of bye spermatozoa considered as membrane-intact cells, except for the slight differences (5 to 5.5%) found at 50 x 106 spermatozoaJmL prefreezing sperm concentration. This could indicate that the cryopreservation process mainly damages acrosome membranes which are less resistant than plasma membranes. In this study, of all the components of the two extenders, the difference in egg yolk concentration is the most hkely to have exerted the best cryoprotectlve effects m Milk-kY extender. It appears that, for the middle preffeezing sperm dilutions (100 to 500 x 106 spermatozoa/mL), the egg yolk preserving effect on plasma and acrosomal integrity (36) is bigger at lower concentrations, as present in Milk-LY extender (range 7.0 to 11.7%, data not shown) compared to Tris-FY extender (range 15.0 to 19 7%, data not shown). At the extreme rate of dilution (,50 and 800 x 106 spermatozoa/mL), Mdk-LY extender did not protect as well as plasma and acrosome membranes of spermatozoa. Motility of spermatozoa at 37 ° C, as well as acrosome integrity, represents a test for evaluating semen quality from the view point of cell functlonahty. The higher levels of post-thaw sperm motility compared to survwal evaluated by Hoechst dye exclusion agree with the findings of Valcarcel et al. (34) in ram and Martemucci et al. (21) m goat spermatozoa. This was attributed to the lower resistance of the plasma membrane to cryodamage than motility organs and nucleus of the cells (31). Post-thaw (0 h) moUlity was improved by Milk-LY extender in prefreezing lower sperm concentrations compared to the Tris-FY extender which, in turn. gave highest sperm concentrations (500 to 800 x l0 t spermatozoa/mL) Mdk-LY extender displays its beneficial effects especially after 3 h incubation where at lower sperm concentrations (50 to 400 x 106 spermatozoa/mL) gives significantly higher percentages of motile spermatozoa compared to Tris-FY At highest sperm concentrations (500 and 800 x 106 spermatozoa/mL) the decrease of sperm motility was less steep in Milk-LY (about 25%) than m Tris (33.5 to 38 7%) reaching similar values. The apparence of improved post-thaw viability after incubation may be a consequence of appropriate higher osmolarity of the Milk-LY extender (450 mOsm), although osmolarity of the Tris-FY (300 to 325 mOsm) was within the range of values tolerated by ram spermatozoa frozen in Tris-based media (200 to 400 mOsm) (32). Similar to our findings, in goat semen hypertonic diluents are well tolerated by spermatozoa at all dilution rates, whereas hypotomc medm are less favorable at high dilution rates and are mainly detrimental at low diluuon rates (27). Contraryto the best in vitro performance of semen frozen m M11k-LY diluent found m this study, in goat semen Azawi et al (2) reported the best restllts with a Tris-fructose egg yolk extender compared to skim-milk egg yolk, with reference to mottlity, viabdity and sperm damage. In vivo experiments did not confirm the superiority of the Milk-LY extender respect to TrisFY Comparison of the resultant fertility show's inconsistent differences between the two extenders observed in vitro that might have been overcome by the deposition of semen in the uterus, a place close to the fertilization site, according to Maxwell and Watson (24). This indicates that either Milk-LY or Tris-FY can be used as extender for cryopreservation of ram semen to be used in laparoscopic insemination because both extenders assure the maintenance after thawing of suitable fertilizing abihty of viable spermatozoa selected by cryopreservation. The overall lambing rates obtained in this study were similar and higher than those reported in earlier studies after cervical insemination with higher doses of semen frozen in either Milk-LY

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extender (33.0 to 75.5%) (6, 7, 30) or Tris-glucose-yolk (30 to 57%) (22, 30) In this study, fertility tended to be higher using the lowest inseminate doses of semen. A fundamental step of manipulation of semen is its dilution performed according to a volume/volume ratio (16, 27) or to a constant concentration of spermatozoa (6, 14, 19), as m thin study. Post-thawing characteristics of spermatozoa were significantly impaired by increase of prefreezing sperm concentration to 800 x 106 spermatozoa/mE Similarly, lambing rates tended to decrease with an inseminate dose of 160 x 106 spermatozoa, corresponding to this final concentration, using either Milk-LY or Tris-FY as extender. The lack of improvement in fertility by increasing the number of spermatozoa inseminated to 160 x 106 spermatozoa/ewe may be a direct consequence of lower post-thaw vtability of spermatozoa frozen at the highest prefreezing sperm concentration (800 x 106 spermatozoa/mL). Furthermore, taking into account that the composition of the diluents was not adjusted for each different sperm concentration, it is possible that it is not adequate for very high densities in cells. Adverse effects of a high sperm concentration also were reported by others who observed a poor post-thaw recovery of spermatozoa for semen diluted to a very low rate (1 : 0.5; semen • diluent) (goat: 27). Semen diluted to higher rates (1:2 to 1 " 23) did not show a difference in motility and fertility (ram: 16; goat: 28), confirming in part our results. In conclusion, Milk-LY extender results in a better in vitro performance compared to Tris-FY when used to dilute semen to intermediate concentrations (100 to 500 x 106 spermatozoa/mL). However, these improvements were not reflected in fertility. The increase of prefreezing sperm concentration to 800 x 106 spermatozoa/mL negatively affects in vitro charactertstics of spermatozoa so that insemination doses, although with a higher sperm number, tended to decrease the lambing rates. Semen doses of 20 to 40 x 106 spermatozoa/ewe were useful when used for laparoscopic insemination. REFERENCES 1. 2. 3 4.

5.

6 7. 8. 9. 10.

Allison A J, Robinson TJ. Fertility of progestagen-treated ewes in relation to the numbers and concentration of spermatozoa in the inseminate. Aust J Biol Sci 1971 ;24:1001-1008. Azawi OI, AI-Dahash SYA, Juma FT Effect of different diluents on Shami goat semen Small Rumin Res 1993;9:347-352 Berndtson WE, Foote RH. The freezabihty of spermatozoa after minimal pre-freezing exposure to glycerol or lactose. Cryobiology 1972;9:57-60. Centola GM, Mattox JH, Burde S, Leary JF. Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using a single-wavelenght fluorescence microscopy Mol Reprod Dev 1990;27:130-135. Cogni6 Y. Current technologies for synchronization and artificial insemination in sheep. In: C.M. Oldham, G.B. Martin, I.W Purvis (eds), Reproductive Physiology of Merino Sheep. The University of Western Australia, 1990;207-216. Colas G Effect of inttial freezing temperature, addition of glycerol and dilution on the survtval and fertilizing ability of deep-frozen ram semen. J Reprod Fertil 1975a;42:277-285. Colas G. The use of progestagen SC9880 as an aid for artificial insemination m ewes. Ann Biol Anim Biochim Biophys 1975b, 15:317-327. Colas G, Guerin Y. A new method for thawing frozen semen. Theriogenology 1981;16:623630. Cross NL, Hanks SE. Effects of cryopreservation on human sperm acrosomes. Human Reprod 1991 ;6:1279-1283. Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome reacted human sperm. Gamete Res 1986; 15:213-226.

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