Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 µM) and retinyl (RI; 0.282 or 2.82 µg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

Retinol improves in vitro oocyte nuclear maturation under heat stress in heifers.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18-21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 µg/ml), retinyl (0.28 µg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

Oestrus cycle characteristics and prediction of ovulation in Catalonian jennies.

The Catalonian donkey breed is in danger of extinction, and much needs to be learned about the reproductive features of its females if breeding and conservation programmes are to be successful. This study reports the oestrous behaviour, oestrus cycle characteristics and dynamic ovarian events witnessed during 50 oestrous cycles (involving 106 ovulations) in 10 Catalonian jennies between March 2002 and January 2005. These jennies were teased, palpated transrectally and examined by ultrasound using a 5 MHz linear transducer-daily during oestrus and every other day during dioestrus. Predictors of ovulation were sought among the variables recorded. The most evident signs of oestrus were mouth clapping (the frequent vertical opening and closing of the mouth with ears depressed against the extended neck) and occasional urinating and winking of the vulval lips (homotypical behaviour). Interactions between jennies in oestrus were also recorded, including mounting, herding/chasing, the Flehmen response, and vocalization (heterotypical behaviour). Nine jennies ovulated regularly throughout the year; one had two anovulatory periods (54 and 35 days). The length of the oestrus cycle was 24.90 +/- 0.26 days, with oestrus itself lasting 5.64 +/- 0.20 days (mean +/- S.E.M.) and dioestrus 19.83 +/- 0.36 days. The incidence of single, double and triple ovulations was 55.66% (n=59), 42.45% (n=45) and 1.89% (n=2), respectively. No significant difference was seen in the number of ovulations involving the left and right ovaries (52.63% [n=70] compared to 47.37% [n=63] respectively; P>0.05). The mean interval between double ovulation was 1.44 +/- 3.98 days. The mean diameter of the preovulatory follicle at day -1 was 44.9 +/- 0.5 mm; the mean growth rate over the 5 days before ovulation was 3.7 mm/day. Data on preovulatory changes in oestrous behaviour, follicle size, follicle texture, the echographic appearance of the follicle and uterus, and uterine tone were subjected to stepwise logistic regression analysis to detect predictors of ovulation. The logit function showed the best predictors to be follicle size, follicular texture and oestrous behaviour. Certain combinations of these three variables allow the prediction of ovulation within 24 h with a probability of >75%.

Effects of freezing/thawing on motile sperm subpopulations of boar and donkey ejaculates.

The main aim of this study is to assess the influence of freeze/thawing on motile sperm subpopulations in ejaculates from two phylogenetically different mammalian species, boar and donkey. Our results indicate that, whereas boar and donkey sperm respond very differently in their mean motion characteristics to freezing/thawing, this process did not change the existence of a 4-subpopulations structure in the ejaculates in either species when these subpopulations were defined by taking values of curvilinear velocity (VCL) as reference. Moreover, the freezing/thawing-linked changes in mean sperm-motion characteristics in both boar and donkey semen were especially due to changes in the proportion among each concrete subpopulation. In this way, the freezing/thawing-induced mean increase in motion characteristics observed in boar sperm was a result of the decrease in the percentage of sperm in Subpopulation 1 (from 53.9%+/-4.7% to 31.2%+/-3.9% after thawing) and a concomitant increase of sperm from Subpopulations 3 (from 13.3%+/-2.5% to 32.6%+/-3.9% after thawing) and 4 (from 3.4%+/-0.9% to 8.0%+/-1.1% after thawing). On the contrary, changes in mean motility of frozen/thawed donkey sperm were linked to an increase in the percentage of sperm in Subpopulation 1 (from 31.5%+/-4.3% to 58.8%+/-4.9% after thawing) and a concomitant decrease of sperm from Subpopulations 3 (from 32.4%+/-3.2% to 6.6%+/-1.8% after thawing) and 4 (from 12.2%+/-2.5% to 7.3%+/-1.9% after thawing). In conclusion, our results seem to indicate that motility changes induced by the freezing/thawing protocol are linked to concomitant changes in both the specific parameters and, more importantly, to the specific percentage of each of the motile sperm subpopulations. These changes did not affect the overall proportion of motile sperm present in both boar and donkey, which is conserved despite the detrimental effect caused by freezing/thawing in both species. Finally, the presence of some kind of motile sperm subpopulations structure has been described in mammalian species with a very great phylogenetic distance, thus suggesting that this structure could play some role in the maintenance of the overall function of mammalian ejaculates.

Pubertal age based on testicular and epididymal histology in Göttingen minipigs.

Göttingen minipigs are used worldwide as nonrodent animal models for toxicologic and pharmaceutical research. Having knowledge of the age for onset of puberty is an important consideration when designing such biomedical experiments. The present study reports an earlier age for puberty on the basis of changes in testicular spermatogenic development in the Göttingen minipig. Testes and epididymides of 24 Göttingen minipigs ranging 5 to 8 weeks of age were obtained for histologic observation after hematoxylin-eosin staining. Microscopic examination was performed to determine the prevalence of cell types (e.g., spermatogonia, primary and secondary spermatocytes, round spermatids, elongated spermatids, luminal sperm) in seminiferous tubules and for the presence of sperm in the cauda epididymis. Puberty was defined as having a majority cell type presence of elongated spermatids in the seminiferous tubules in conjunction with the presence of spermatozoa in the cauda epididymis. Puberty was identified in two males at 6 weeks of age, two males at 7 weeks of age, and in all 6 males at 8 weeks of age. In conclusion, the age of puberty in male Göttingen minipigs occurs by 8 weeks of age, an earlier age than previously reported.

Survival Time of Cross-Match Incompatible Red Blood Cells in Adult Horses.

BACKGROUND: There is a markedly reduced half-life of transfused RBCs when donor and recipient cats or humans are cross-match incompatible. Only 10-20% of horses have naturally occurring alloantibodies. Therefore, cross-match testing before blood transfusion is not always performed. HYPOTHESIS: Cross-match incompatibility predicts shortened RBC survival time as compared to that of compatible or autologous blood. ANIMALS: Twenty healthy adult horses. METHODS: Prospective trial. Blood type, anti-RBC antibody screen (before and 1 month after transfusion) and major and minor cross-match determined 10 donor-recipient pairs. Two pairs were cross-match compatible, the remainder incompatible. Donor blood (4 L) was collected into citrate phosphate dextrose adenine-1, labeled with NHS-biotin, and transfused into recipients. Samples were collected at 1 hour and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after transfusion, and biotinylated RBCs were detected by flow cytometry. Horses were monitored for transfusion reaction during transfusion and daily for 5 days. RESULTS: Cross-match incompatibility was significantly associated with decreased RBC survival time (P < .001). The half-life of transfused incompatible (cross-match >1+) allogenic equine RBCs was 4.7 (95% CI, 3.2-6.2) days versus 33.5 (24-43) days for compatible pairings. Cross-match incompatibility was associated with acute febrile transfusion reaction (P = .0083). At day 30, only 1 horse had developed novel anti-RBC antibodies. CONCLUSIONS AND CLINICAL IMPORTANCE: Cross-match incompatibility was predictive of febrile transfusion reaction and shortened transfused RBC survival, but did not result in production of anti-RBC antibodies at 30 days. Cross-match testing before transfusion is recommended.

Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

Retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro.

An experiment was designed to study the interaction between fresh/frozen-thawed donkey spermatozoa and zona pellucida (ZP)-free bovine oocytes in an attempt to develop a model for assessing cryopreserved Catalonian donkey sperm function. Semen from five donkeys was collected using an artificial vagina. Sperm motility and viability were immediately assessed and the semen sample cryopreserved. Sperm viability and motility were then reassessed immediately after thawing. The motion characteristics of the fresh and frozen-thawed spermatozoa were determined using a computer-assisted sperm analysis system. In vitro-matured cow oocytes were inseminated with different percent live donkey sperm (high (>60%) or low (<40%) viability donkey sperm). After 18h of co-incubation, the oocytes were fixed, stained with 4',6-diamidino-2-phenylindole (DAPI) and examined for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. Frozen-thawed spermatozoa from high viability semen showed significantly lower VCL, VAP and mean ALH values than did high viability fresh spermatozoa. In contrast, frozen-thawed spermatozoa of low viability had significantly higher velocity values than fresh spermatozoa of low viability. A significant positive correlation (P<0.01) was detected between percentage fertilization and viability (r=0.84), and between percentage fertilization and certain CASA parameters (VAP, r=0.56; VCL, r=0.61 and mean ALH, r=0.68). Fresh or frozen-thawed high viability spermatozoa penetrated 90.1% and 85.4% of bovine oocytes respectively. Lower rates of penetration were observed for fresh and frozen-thawed low viability spermatozoa (34% and 22.5% respectively). The donkey spermatozoa were able to fuse with the oolema and even to decondense and form the male pronucleus (85-94%). Larger numbers of penetrated spermatozoa per oocyte were recorded when high viability sperm samples were used, whether fresh (3.02 vs. 1.12 for low viability sperm) or frozen-thawed (3.41 vs. 1.47). Consequently, low viability sperm samples showed higher percentages of monospermic penetration (91.17% and 61.97% for fresh and frozen-thawed sperm samples respectively). These findings suggest that bovine oocytes provide a useful model for assessing the penetration potential of frozen-thawed donkey sperm.

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen.

The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25x10(6) sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 degrees C, aliquots of these semen samples were incubated at 37 degrees C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns. The initial sperm concentration had a significant (P<0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live spermatozoa in the fresh semen significantly influenced the number of spermatozoa damaged by centrifugation, but not centrifugation efficacy.