Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents

The addition of components of animal origin (egg yolk, milk) to most commercial diluents used to freeze bull semen represents a potential risk of contamination of the doses with bacteria or mycoplasma. A series of quantitative and qualitative analyses were performed to detect microbiological contamination observed in Biociphos plus (a new lecithin-glycerol based freezing salt buffer), in an egg yolk diluent (Triladyl) or in an egg yolk + milk-based (Laiciphos) diluent of bull semen. The 2 diluents containing animal products showed moderate (10 to 60 CFU/mL) contamination (17/17 samples) with bacteria or mycoplasma, or both, while no contamination was observed in the 6 examined batches of Biociphos plus. Biociphos plus was also compared with another commercial diluent (Laiciphos) for use in freezing bull semen intended for in vitro and/or in vivo fertilization. No difference (P > 0.05) could be detected between the 2 diluents for in vitro fertility rates (percentage of cleaved zygotes: 85.7% and 88.0%, respectively, for Laiciphos and Biociphos plus). Similarly, 2 series of comparisons conducted in dairy cows artificially inseminated with semen frozen in either Biociphos plus or Laiciphos showed no difference in fertilizing capacity (tested at 60 to 90 d; P > 0.05) irrespective of the age of the bulls (Trial 1, bulls aged 14 to 15 m.o.; Trial 2, bulls aged 2 to 5 yr, field trials). It is concluded that, in addition to maintaining the fertilizing capacity of bull semen at levels comparable to those observed with standard freezing diluent, Biociphos plus also prevents microbiological contamination by bacteria or mycoplasma, both of which are generally present in the various commercially available sources of egg yolk.     

Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field.     

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.     

Induction of immunity to spermatozoa in male domestic fowl and effects on fertility.

Male fowl were immunized intravenously (i.v.) or intramuscularly (i.m.) with spermatozoa to assess the effects of immunity to spermatozoa on fertility. Histological and immunofluorescence evaluations of testis and ductus deferens tissues after 24 weeks of immunizations revealed immune cell infiltration and immunoglobulin associated with spermatozoa. The long-term immunization regimen resulted in significant antisperm antibody titres in the immunized groups. When semen from i.v.-immunized males was used to inseminate females, fertility over 7 days was reduced (P less than 0.05). A subsequent experiment using a 10-week i.v. immunization scheme also led to high antisperm titres. Spermatozoa from these males were characterized by lower fertility and duration of fertility than those of controls (P less than 0.05). As in mammals, a reduction in fertility may result from exposure of avian males to sperm antigens.     

Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage.

Motility characteristics of turkey spermatozoa before and after storage for 24 h at 7 degrees C in diluent with and without bovine serum albumin (BSA; 1% final concentration) were measured by computer-assisted semen analysis. BSA significantly increased the percentage of motile spermatozoa and sperm velocity, linearity, lateral head displacement and beat frequency in each treatment, but BSA in fresh or stored semen in diluent did not augment hen fertility over 15 weeks of egg production. Fatty-acid-free BSA, globulin-free BSA and Fraction V BSA all significantly increased each sperm motility characteristic compared with semen in diluent alone. The lack of correlation between sperm motility and fecundity emphasizes the need to develop procedures for semen evaluation that accurately predict the fertilizing capacity of an aliquot of semen.     

Comparison of three diluents for the storage of fresh bovine semen

In New Zealand, 95% of the semen used for artificial insemination in cattle is processed as liquid semen. Storage of liquid semen for up to 3 days in Caprogen) diluent enables a 10-fold reduction of the insemination dose, compared to frozen-thawed semen, without a reduction in fertility. In this Caprogen) diluent spermatozoa are stored under N2 gas in the presence of catalase. However, a new diluent (CEP-2), which was originally based on the biochemical composition of bovine cauda epididymal plasma, could become an appropriate alternative to Caprogen. In this study, the effect of addition of catalase to bovine spermatozoa stored for 6 days in CEP-2 diluent under aerobic and anaerobic conditions was evaluated and compared with a Tris diluent. Additionally, the quality and in vitro fertilizing capacity of fresh bovine semen stored for 6 days at 5 degrees C in the Triladyl, CEP-2 (without catalase and N2 gas) and Caprogen diluent were compared. Addition of 4.5 mg/mL catalase to CEP-2 diluent under aerobic and anaerobic conditions had no effect on sperm quality. Spermatozoa stored in CEP-2 diluent moved faster and straighter than spermatozoa stored in Triladyl or Caprogen diluent. The in vitro fertilization and polyspermy rates did not differ significantly between spermatozoa stored for 6 days at 5 degrees C in CEP-2 and Caprogen diluent, but were significantly lower for spermatozoa stored in Triladyl diluent. We can conclude that based on the in vitro results, the CEP-2 diluent is a better diluent than Triladyl and a good alternative to the Caprogen diluent for long term storage of fresh bovine semen at 5 degrees C. To confirm these promising in vitro results further in vivo experiments are required.     

Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

Equine frozen semen: freezability and fertility field results

The freezability of stallion semen defined as the number of selected ejaculates/total number of ejaculates frozen from 161 different stallions was analyzed. Of the stallions, 19, 30, 27 and 24% had a freezability of 0%, 0 to 33%, 33 to 66%, over 66%, respectively In 85 different stallions, the correlation of freezability between first and second year was 0.60 (P < 0.001). The relationship between fertility with fresh and frozen semen and freezability was analyzed in 40 stallions whose freezability and fertility information was recorded during 5 years. There was a strong relationship between fertility of fresh semen and semen freezability (P < 0.001). However, the relationship between fertility of frozen semen and freezability was not as marked (P < 0.05). Analysis of the field fertility per cycle results when mares were bred with 300 or 150 x 10(6) total spermatozoa at different frequencies until ovulation indicated that mares that were inseminated 2 times or more per estrus show an improved fertility in comparison with mares inseminated only once (34%, n = 1576 vs 26%, n = 626; P < 0.001). Foaling rate when mares were inseminated with frozen semen (1858 mares during 8 breeding seasons) was mainly influenced by mare age (< 16 years: 54% vs >/= 16 years 42% p < 0.001). Date of first insemination (before May 15: 58% vs after May 15: 37%) also had a significant effect on foaling rate (P < 0.001).     

The effect of buffer osmolality on the survival of cat (Felis catus ) spermatozoa at 5 degrees C

Cat semen was diluted at 37 degrees C in Tes-Tris buffer (TesT), pH 7.5, at osmolalities ranging from 195 to 390 m0sm/kg, cooled to 5 degrees C over 90 minutes and stored for 24 hours at that temperature. Motility and percentage of spermatozoa staining with a supravital stain were estimated before cooling, after cooling and after storage for 24 hours. The osmolality of undiluted pooled ejaculates from five animals was measured, and also that of different diluents (citrate with phosphate buffer, lactose and TesT-egg yolk) used for cat semen. The osmolality measurements of cat semen suggested an osmolality of less than 320 m0sm/kg at ejaculation, increasing with time after ejaculation. Varying the egg yolk concentrations (2% to 20%) did not affect the osmolality of TesT diluent. Diluent osmolalities of less than 292 m0sm/kg were found to reduce sperm motility significantly (P <0.001 ) although there was no significant increase in the percentage of cells staining with a supravital stain, while those greater than 325 m0sm/kg increased the variation of response among animals. Cooling and storage significantly reduced motility (P <0.01 to P <0.001 ) and increased the number of stained cells (P <0.001 ). There were significant differences between ejaculates (P <0.01 ) and significant interactions between osmolality and cooling/storage (P <0.05 to P <0.001 ). The best overall results were seen with a TesT diluent of 292 to 325 m0sm/kg which supported good motility for at least 24 hours.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Testicular development, ultrasonographic and histological appearance of the testis in ram lambs immunized against recombinant LHRH fusion proteins

Sixteen native ram lambs weaned at 10 wk of age were divided into two groups. Eight animals were immunized against LHRH with a mixture of two fusion proteins: ovalbumin-LHRH-7 and thioredoxin-LHRH-7. The immunized lambs received a primary immunization plus two booster immunizations at 4 and 12 wks. Animals in the control group (n=8) were not treated. Scrotal measurements and blood samples were taken at 2-week intervals. Beginning at 25 wk of age, semen was collected and sexual behaviour was evaluated on a weekly basis. At 35 and 37 wk of age testes and accessory glands of all animals were subjected to ultrasound scanning. At 37 wk of age animals were slaughtered and testes were evaluated histologically. Serum LHRH antibodies (P<0.01) were detected in animals of the immunized group which had reduced serum testosterone concentrations (P<0.01). Testicular development was suppressed in the immunized animals (P<0.01). Immunized animals exhibited mounting activity 5 wks later than control animals. No mature spermatozoa containing ejaculates were collected from immunized animals. Control animals had moderately echogenic ultrasonographic appearance at 37 wk age, whereas immunized animals had hypoechogenic images. Mean seminiferous tubule diameter in immunized lambs was significantly smaller than that in control lambs. Basal membrane was thickened and hyalinized; there was an increase in peritubular connective tissue. No proliferating spermatogonia or mature spermatozoa were present in the tubules in these animals. There were no differences in the ultrasonographic appearance of prostate and vesicular gland between control and immunized animals. The LHRH recombinant fusion proteins were effective in immunological castration in ram lambs when started at 10 wk of age as noted by differences in serum testosterone, testicular histology and ultrasonographic appearance of testis and weight of accessory sex glands. Determining the effects of immunization on ultrasonographic appearance of the testis related to time after immunization requires further investigations.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Fertility test of frozen boar semen.

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0-25 X 10(9)/ml sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1-0 X 10(9)/ml sperm concentration, and extended wtih FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8. The mean farrowing for the 32 animals inseminasted was 34-4%. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5 degrees C were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37-5% farrowed.     

Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen

The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio? cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio? (BC) or Botu-Crio? which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment 1, sperm frozen in BC and BCLS45 exhibited similar (P>0.05) percentages of total motile sperm (61% and 61%, respectively); progressively motile sperm (27% and 27%, respectively) and sperm with intact plasma membranes (IMP; 53% and 57%, respectively). Similarly, sperm frozen in BC or BC containing any concentration of soybean lecithin maintained similar (P>0.05) percentages of total motile sperm (61-68%) and progressively motile sperm (27-31%). In the first fertility trial, we used cryopreserved semen from a single stallion was inseminated into mares. The semen from the sperm that were frozen in BC diluent resulted in a higher fertility rate (66%, 16/24) compared to the sperm that were frozen in BCLS45 diluent (17%, 5/29; P<0.01). Similarly, in a second fertility trial, the mares that were inseminated with the sperm that were frozen in BC diluent exhibited a higher fertility rate (66%, 16/24) compared to the mares that were inseminated with the sperm that were frozen in BCLS20 (40%, 10/25; P<0.05). Finally, in a third trial, the sperm that were frozen in BC resulted in a higher fertility rate in mares (75%, 18/24) compared to the sperm that were frozen in BCLS10 (41%, 10/24; P<0.05). Although replacing the egg yolk in the BC cryodiluent with soybean lecithin provided similar laboratory results for stallion sperm, after cryopreservation, the sperm that was frozen with soybean lecithin in the diluent correlated with lower fertility rates. Based on these results, we concluded that the use of BCLS can be used as an alternative diluent for cryopreserving stallion sperm. However, the resulting reduced fertility rate is a matter of concern. Further studies are necessary to clarify the reasons for this decrease in fertility and to determine the optimal lecithin concentration for diluents to freeze stallion sperm.     

Relationship between an 85 kDa protein and the protective effects of Mycoplasma pneumoniae.

In the immunoblot analysis, sera from patients infected with Mycoplasma pneumoniae reacted with the 168 kDa (P1) and the 85 kDa proteins of virulent strain FH-P24 and P24-S1 mutant strain but not with the 85 kDa protein of P24-S11. Sera of hamsters and BALB/c mice, which had been immunized with live vaccines, were tested. In FH-P24 immunized animals, 100% or 80%, and in P24-S1, 40% of hamsters and 60% of BALB/c mice, developed antibodies against the 85 kDa protein, but antibodies were not detected in sera of P24-S11 immunized animals. The correlation between the development of antibodies to 85 kDa protein in the sera of vaccinated animals and the effects of protection by living vaccines were suggested.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Birth of the first mithun ( Bos frontalis) calf through artificial insemination

The study describes the standardization of a suitable semen cryopreservation protocol for the first time in mithun (Bos frontalis) and birth of the first mithun calf through artificial insemination. The semen samples were collected from adult bulls through the rectal massage method and cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluent. The diluted semen samples were packaged in 0.50 ml straws and kept at 5°C for 4 h for equilibration. Following the equilibration, the straws were frozen into liquid nitrogen vapour for 10 min and then plunged into liquid nitrogen for storage. It was observed that the progressive motility (%) decreased significantly (P < 0.01) in cryopreserved semen (43.3 ± 4.1) compared with fresh samples (76.6 ± 3.3). The percentages of live spermatozoa (P < 0.01) and spermatozoa with intact acrosome (P < 0.05) also decreased significantly in cryopreserved semen (54.0 ± 3.3 and 64.6 ± 5.3) compared with fresh samples (79.3 ± 2.6 and 85.3 ± 1.8). Simultaneously, the total morphological abnormality (%) was found to be significantly (P < 0.01) higher in cryopreserved samples (15.46 ± 2.68) than in fresh semen (3.85 ± 0.63). A total of three mithun cows were inseminated using the cryopreserved semen. All the cows conceived following insemination and gave birth to healthy calves. The study revealed that mithun semen can be cryopreserved efficiently using tris-egg yolk-glycerol diluent, which can be further used for artificial insemination.     

Centrifugation of stallion semen and its storage in large volume straws.

In a study of different methods of handling stallion semen for deep freezing, ejaculates were divided into 3 portions, the first of which was diluted 1:2 with lactose--egg yolk--glycerol diluent and frozen in pellet form. The second aliquot was centrifuged without any diluent and the third portion was initially diluted with an experimental diluent (Merck) and then centrifuged for 5 min at 1000 g. The second and third portions were frozen in large volume straws each of which contained one whole insemination dose of 1 or 2 X 10(8) progressively motile spermatozoa. The addition of a diluent to the semen before centrifugation and freezing (portion 3) resulted in an increase in sperm motility after thawing. Motility was further increased by the use of a recently developed diluent after centrifugation and before freezing. In one fertility trial, 12 of 19 mares (63%) conceived following a single insemination of frozen semen during one oestrous period.     

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.