Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not

A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and horse semen was either used immediately, or cooled according to some steps of the pre-freezing procedure or frozen and thawed. The pre-freezing procedure included semen dilution, centrifugation, resuspension in milk or in INRA82+2% egg yolk+various % CPA (expressed as final concentrations in extended semen (v/v)) and then cooling to 4 degrees C. PC was similar in mares and jennies inseminated with donkey semen cooled to 4 degrees C in milk. However, the PC was significantly higher in mares than in jennies when donkey semen was frozen with 2.2% glycerol (36%, n=50 cycles vs. 11%, n=38 cycles; P<0.01). Increasing the concentrations of glycerol (0, 2.2, 3.5, 4.8%) before cooling stallion semen resulted in a progressive decrease in mare PC (87, 53, 53, 13% (n=15 cycles for each concentration); P<0.0001). The addition of 2.2% glycerol before cooling donkey semen decreased the PC measured in jennies to 0. The replacement of glycerol by 2% dimethylformamide increased the fertility obtained in jennies with cooled donkey semen (PC: 67%, n=12 cycles) but did not increase the fertility obtained with frozen-thawed donkey semen (PC: 11%, n=28 cycles with dimethylformamide vs. 0%, n=16 cycles with glycerol). In conclusion, this study clearly shows that the ability of jennies to conceive after AI with donkey frozen semen is lower than that of mares. Glycerol affects the fertility of donkey and stallion spermatozoa as early as during the pre-freezing procedure. In consequence, the glycerol level must be low in frozen equine semen to provide good fertility. The toxic dose of glycerol for donkey spermatozoa seems to be almost half that for stallion spermatozoa. Whether this greater sensitivity of donkey spermatozoa to glycerol is responsible for the low success of semen cryopreservation in jennies is not so obvious because replacement of glycerol by dimethylformamide was not much more effective in terms of fertility.     

Sexual behavior in ovariectomized and seasonally anovulatory pony mares (Equus caballus)

Ten ovariectomized (OVEX) and ten intact, but seasonally anovulatory (ANOV), pony mares were observed for sexual activity with five stallions, using a “harem group” social testing paradigm (two OVEX and two ANOV mares plus one stallion per group) for 15 consecutive daily tests lasting 20 min each. All mares in both conditions showed proceptive behavior in at least one test, all mares but one were mounted, and 14 of 20 mares received ejaculations. No statistical differences were found between the two conditions for any measure of proceptivity, copulatory activity, or days in estrus. The quality of estrus was judged to be equivalent to that displayed by periovulatory mares during their initial and terminal days of estrus, but less intense than that seen near ovulation. Mares in both groups were in estrus during approximately 60–70% of the tests and only 3 of the 20 mares were sexually refractory for more than five consecutive tests. Thus, the typical 2-week phase of sexual refractoriness seen in intact diestrous mares was absent in OVEX and ANOV mares, suggesting that the ovary plays a major role in actively suppressing estrous responses during the luteal phase of the cycle.     

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P = 0.0201) in BoHV-FBS (80.4 ± 8.9%; mean ± SD) compared with that of noninfected oocytes (89.9 ± 6.5%); neither differed from BoHV-PVA (87.3 ± 7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5 ± 7.5% vs. 92.2 ± 5.5%, control vs. infected) and development of embryos (41.7 ± 9.9% vs. 44.3 ± 7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6 ± 10.3% vs. 40.8 ± 9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P = 0.1462, P = 0.5402, and P = 0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P = 0.0140 to M/B/EB and P = 0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6 ± 4.6 vs. 25.0 ± 5.5 and 31.6 ± 4.6 vs. 20.2 ± 5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.     

Efficiency of superovulation and in vivo embryo production in eFSH-treated donor mares after estrus synchronization with progesterone and estradiol-17β

Reliable methods of regulating estrus and stimulating superovulations in equine embryo transfer programs are desirable. Our objectives were to investigate the efficacy of a progesterone and estradiol-17β (P&E) estrus synchronization regimen in mares with and without subsequent equine follicle-stimulating hormone (eFSH) treatment and to examine the effects of eFSH on folliculogenesis and embryo production. Cycling mares were treated with P&E daily for 10 d. On the final P&E treatment day, prostaglandin F_2α was administered, and mares were randomly assigned to one of two treatment groups (n = 20 mares/group). In both groups, mares were examined daily by transrectal ultrasonography. In the eFSH group, twice-daily eFSH treatments were initiated at follicle diameter 20 to 25 mm and ceased at follicle ≥35 mm; human chorionic gonadotrophin (hCG) was administered after 36 h. In the control group, eFSH treatments were not given, but hCG was administered at follicle ≥35 mm. Mares were inseminated with fresh semen, and embryo recovery attempts were performed 8 d postovulation. Synchrony of ovulations within each group appeared to be similar. Six mares in the eFSH group failed to ovulate. The eFSH treatment resulted in higher (P < 0.05) numbers of preovulatory follicles and ovulations; however, embryo recovery rate did not increase (eFSH 1.0 ± 0.4 vs. control 0.95 ± 0.1 embryos/recovery attempt), and embryo per ovulation rate was significantly lower (36% vs. 73%). The eFSH-treated mares had significantly higher frequency of nonovulatory follicles (28% vs. 0) and higher periovulatory serum concentrations of estradiol-17β. Based on our findings, combined P&E and eFSH regimens cannot be recommended for cycling donor mares.     

Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies

The aims of this study were (1) to evaluate motility parameters of donkey jack (jack; Equus asinus) semen cryopreserved in INRA-96 (INRA; IMV Technologies, France, 2% egg-yolk enriched) using either glycerol (GLY) or ethylene glycol (EG) as a cryoprotector; (2) to compare in vitro the postthaw re-extension with homologous seminal plasma (SPL) or INRA; (3) to compare fertility in donkey jennies (jennies; Equus asinus) timed artificially inseminated with jack semen cryopreserved using GLY or EG, re-extended with INRA; (4) to compare fertility in jennies timed artificially inseminated with jack semen cryopreserved using GLY re-extended with SPL, INRA, or not re-extended (NN); and (5) to describe some preliminary results of the inflammatory uterine response postbreeding. Semen from two jacks was collected and frozen in an INRA-2% egg yolk extender added of either 2.2% GLY or 1.4% EG. Postthaw motility was evaluated by a computer-assisted motility analyzer. Uterine inflammatory response and fertility were evaluated after artificial insemination (AI) of 13 jennies with frozen-thawed semen, either further extended with INRA (Group GLY-INRA, 13 cycles, and EG-INRA, 8 cycles), or with SPL (Group GLY-SPL, 13 cycles), or not re-extended (GLY-NN, 5 cycles). In each cycle, jennies were bred twice with 500 × 10⁶ sperm cells (250 × 10⁶ from each jack), at fixed times after induction of ovulation, and uterus was flushed at 6 and 10 h after first and second breeding, respectively. Cells in the recovered fluid were counted and distinguished as polymorphonuclear neutrophils (PMN) or other cell types. Total and progressive motility did not differ between cryoprotectants, but were higher when semen samples were re-extended in INRA, compared with SPL (P < 0.05). Pregnancy was diagnosed by transrectal palpation and ultrasonography examinations at 14 and 16 days postovulation. In 7/13 (53.8%) jennies and 12/39 (30.4%) cycles postbreeding intrauterine fluid accumulation was observed, with no differences between treatments (P < 0.05). Polymorphonuclear neutrophil numbers and concentrations were higher in the first flushing compared with the second, and PMN concentration was higher in GLY-SPL than in GLY-INRA (P < 0.05). Pregnancy rates in GLY-SPL, GLY-INRA, EG-INRA, and GLY-NN were 8/13, 3/13, 2/8, and 1/5, respectively. There was no significant difference either between the two cryoprotectants re-extended in INRA, or between re-extension groups. There was however a trend for GLY-SPL to improve pregnancy rates compared with GLY-INRA (P = 0.055). These results indicate that it is possible to obtain similar postthaw sperm motility and pregnancy rates using GLY or EG as a cryoprotectant for donkey semen, and that in the conditions of this study the re-extension in SPL of thawed semen before AI showed a trend toward the improvement of fertility and increased PMN concentration in uterine flushings.     

The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: A field study

The association between use of hormone treatments to induce estrus and ovulation and the incidence of hemorrhagic anovulatory follicles (HAFs) was studied in a mixed population of mares (Equus caballus) during two breeding seasons in a commercial breeding clinic. Mares treated with cloprostenol (CLO) were more likely to develop HAFs than were mares with spontaneous cycles (P < 0.001) or those treated with human chorionic gonadotropin alone (P = 0.08). There was no significant effect of season on the incidence of HAFs. The mean (±SEM) interval from CLO treatment to beginning of HAF development was 6.1 ± 0.5 d. Age of mares with HAF cycles was not different (12 ± 1.3 yr; P > 0.05) from that of mares with ovulatory cycles (10.5 ± 1.5 yr).     

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 × 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 ± 2.3 d postpartum, received a 3 mg norgestomet ear implant sc, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 μg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means ± SEM, 1.53 ± 0.1 vs. 0.48 ± 0.1 mm/d; P < 0.0001), its diameter on Day 11 (11.4 ± 0.6 vs. 9.3 ± 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 ± 1.1 vs. 71.1 ± 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Use of two anesthetic combinations for semen collection by electroejaculation from captive coatis (Nasua nasua)

The objective was to compare the effects of ketamine-xylazine or tiletamine-zolazepam combinations as anesthetic protocols for captive coatis (Nasua nasua) for semen collection by electroejaculation. Five mature male coatis were physically restrained and then anesthetized by im injections of ketamine (10 mg/kg) plus xylazine (1 mg/kg) or a tiletamine-zolazepam combination (8 mg/kg). For the two combinations, additional quarter-doses of ketamine or the tiletamine-zolazepam combination were administered when necessary. Semen was collected by electroejaculation and immediately evaluated for color, volume, pH, sperm motility, vigor, morphology, acrosomal integrity, and percentage of live cells. Overall, collection of nine ejaculates was attempted from five animals for each treatment. Regardless of the anesthetic combination, all animals developed an erection during each attempt to collect semen. Ejaculates were obtained in all (9 of 9) attempts that used ketamine-xylazine for anesthesia, but in only 3 of 9 attempts (P < 0.05) when tiletamine-zolazepam was used. All ejaculates contained sperm, with no significant differences in semen characteristics between the two anesthetic combinations. Recovery was smooth in all animals. In conclusion, semen collection by electroejaculation in coatis was significantly more successful with the use of a ketamine-xylazine combination than with tiletamine-zolazepam.     

Sexual behavior following introduction of a stallion into a group of mares

A rested stallion was introduced daily for 30 days into each of three herds of 20 mares. Observations of sexual and mating behavior were made for one hour. The stallion remained with a herd until another stallion was introduced the following day. Other mares were isolated from stallions and were bred by artificial insemination. The diameter or growth rate of the preovulatory follicle for the six days preceding ovulation and the length of the interovulatory interval for mares which did not become pregnant were not affected significantly by the presence of a stallion. The number of breedings per hour of observation (2.4 ±0.2) and the length of the interval from introduction of a stallion into the herd of mares to first breeding (12 ±1 min) were significantly different among stallions, but the length of the interval between breedings (17 ±2 min) was not. The mean number of breedings per stallion per hour was not affected significantly by the number of posturing (estrous) mares. The number of times that the stallion rebred the same mare when more than one mare postured during the observation hour (49%) was greater (P<0.01) than what would be expected to occur by chance (30%). The hypothesis that breeding occurs preferentially in those estrous mares that are closest to ovulation was not supported, except for significantly lower breeding activity in posturing mares on days 8 and 7 before ovulation and on days 0 (day of ovulation) and ?1 (26% bred) than on days 6 to 1 (52%).     

Assessment of pregnancy in the late-gestation mare using digital infrared thermography

The objective of this study was to investigate use of digital infrared thermal imaging (DITI) to determine whether surface temperature gradient differences exist between pregnant and nonpregnant mares as a noncontact method to determine pregnancy status. On the day measurements were collected, each pregnant mare (n = 10; beginning at 292.4 ± 1.4 d of gestation) was paired with a nonpregnant mare (n = 17). Ambient temperature, DITI measurements (left and right flank, wither temperatures [i.e., animal surface control] and background temperature), and rectal temperatures were obtained every 7 d for 5 wk before parturition and for 3 wk after parturition. There were no differences (P > 0.10) in temperature of the left and right side within groups; therefore, data were pooled. Pregnant mares had a higher (P < 0.01) flank temperature than that of nonpregnant mares (36.0 ± 0.2 °C vs. 34.2 ± 0.2 °C, respectively). Moreover, the difference (2.4 °C) in flank temperatures between the pregnant and nonpregnant mares was greater when the ambient temperature was <19 °C. Flank and wither temperatures were positively correlated (R = 0.72; P < 0.01) and were positively correlated with ambient temperature (R = 0.48 and 0.64, respectively; P < 0.01). However, wither temperatures (skin control site) did not differ (P > 0.10) between pregnant and nonpregnant mares. In conclusion, late-gestation mares had higher flank temperatures than those of nonpregnant mares, regardless of environmental conditions, however discriminating abilities were greater when ambient temperature was lower. We inferred that DITI may have value in confirming mid- to late-gestation pregnancies in some species by noncontact means, as observed in the mare.     

Association of the rs6235 variant in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene with obesity and related traits in a Taiwanese population

One particularly interesting single nucleotide polymorphism (SNP), rs6235 (encoding an S690T substitution), in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene has been widely associated with obesity in several European cohorts. The present study was intended to investigate the association between the PCSK1 rs6235 SNP and the prevalence of overweight or obesity, or obesity-related metabolic traits in a Taiwanese population. A total of 964 Taiwanese subjects with general health examinations were analyzed. Our data revealed no association of PCSK1 rs6235 with the risk of obesity or overweight in the complete subjects. However, the PCSK1 rs6235 SNP exhibited a significant association with overweight among the male subjects (P = 0.03), but not among the female subjects. Furthermore, the carriers of GG variant had a significantly higher waist circumference than those with the CC variant (82.5 ± 11.5 vs. 81.2 ± 10.2 cm; P = 0.01) and those with the CG variant (82.5 ± 11.5 vs. 81.4 ± 10.4 cm; P = 0.021). In addition, the carriers of GG variant had a higher diastolic blood pressure than those with the CC variant (81.9 ± 14.2 vs. 80.3 ± 12.9 mm Hg; P = 0.023). Our study indicates that the PCSK1 rs6235 SNP may contribute to the risk of overweight in men and predict obesity-related metabolic traits such as waist circumference and diastolic blood pressure in Taiwanese subjects.     

Induction of male sex behavior in pony mares with testosterone propionate

Two pony mares were administered 150 mg of testosterone propionate every other day for 20 days (ten injections) and every ten days there-after. An additional two mares and one stallion were not treated and served as controls. Testosterone propionate was dissolved in absolute ethanol and administered subcutaneously. Sex behavior tests were conducted 26 and 40 days after the first injection. Control mares exhibited very little male sex behavior. Both testosterone propionatetreated mares, however, exhibited mounting, sniffing, flehmen, biting and vocalization behavior in the presence of an estrous mare. The testosterone propionate-treated mares mounted and bit estrous mares more frequently than the stallion but exhibited less sniffing, flehmen and vocalization behavior in the presence of an estrous mare than the stallion. Testosterone propionate-treated mares and the stallion mounted an estrous mare 23.3 +/- 9.7 seconds and 172.5 +/- 22.5 seconds, respectively, after being introduced into the pen. Mares in estrus were mounted by the testosterone propionate-treated mares and the stallion an average of 4.0 +/- 1.3 and 1.0 +/- 0 times, respectively, during a ten-minute test. None of the non-estrous mares was ever mounted by the testosterone propionate-treated mares. In summary, testosterone propionate induced male sex behavior in intact mares and the testosterone propionate-treated mares effectively detected estrous mares.     

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.     

Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition

The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle ≥25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n = 18) were treated twice daily with eFSH (12.5 mg im) until they achieved a follicle ≥35 mm; hCG was given 36 h later. In the deslorelin group, mares (n = 18) were treated twice daily with deslorelin (63 μg im) until a follicle ≥35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2 d (7.3, 8.9) and 7.2 d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean ± S.E.M.; 3.4 ± 0.4 vs. 1.1 ± 0.1 ovulations), and more embryos were recovered (2.6 ± 0.5 vs. 0.4 ± 0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas)

The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (±SD) weight of female toads producing eggs (58.9 ± 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 ± 7.0 g). Similarly, four of seven nonhibernated females (58.8 ± 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment.     

Effects of different extenders and centrifugation/washing on postthaw microscopic-oxidative stress parameters and fertilizing ability of Angora buck sperm

The main objective of the current study was to evaluate the effects of extender type and centrifugation/washing prior to cryopreservation on the postthaw sperm parameters, lipid peroxidation, and superoxide dismutase activity of Angora buck (Capra hircus ancryrensis) sperm. Ejaculates collected from three Angora bucks were used in this study. Two consecutive ejaculates from each buck were pooled and split into equal parts in four Falcon tubes. Two tubes were diluted at 37 °C and then centrifuged to remove semen plasma. After centrifugation, two sediment parts were diluted with a Tris-based extender and commercial Bioxcell extender, respectively. The remaining two parts, which were not centrifuged/washed, were diluted with the above-mentioned extenders, respectively. Diluted samples were cooled to 5 °C and frozen in 0.25-mL French straws to be stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 sec in a water bath for evaluation. The semen part with centrifugation/washing in the Bioxcell extender (BC) demonstrated a higher rate of subjective motility (58.1 ± 3.0%) compared with that of groups with (TC) or without (T) centrifugation/washing in the Tris-based extender (P < 0.01). Angora buck sperm frozen with (BC) or without (B) centrifugation/washing in the Bioxcell extender demonstrated higher percentages of motility (60.6 ± 2.7% and 54.3 ± 4.8%, respectively) compared with that of groups T and TC. The postthaw progressive motility rate (22.3 ± 2.7%) was significantly greater for semen parts diluted in B compared with that of other groups. BC gave rise to a lower value of average path velocity (90.0 ± 5.2 μm/sec) compared with that of other groups (P < 0.01). For straight linear velocity and linearity index, the highest values (103.2 ± 4.7 μm/sec, 47.5 ± 1.6% and 94.8 ± 3.0 μm/sec, 44.8 ± 1.1%, respectively) were obtained from B and TC (P < 0.001). For sperm acrosome and total abnormalities, TC gave the highest values (11.2 ± 0.6% and 26.6 ± 1.5%, respectively, P < 0.01). In the group frozen in BC, the percentage of membrane integrity assessed by hypo-osmotic swelling test was higher (61.2 ± 2.2%) than that of the other groups (P < 0.001). With respect to fertility results based on 35-d pregnancy rates, BC gave a higher rate (76.5%) than that of TC (27.8%, P < 0.05). Malondialdehyde formation was found to be lower (1.64 ± 0.26 nmol/L) in BC than in the other groups after the freeze-thawing process (P < 0.001). In the semen part frozen in BC, superoxide dismutase activity was higher (0.18 ± 0.02 U/mg protein) compared with that of the other groups (P < 0.05). Further studies are required to obtain more precise results for the characterization of oxidative stress parameters and fertilizing ability in cryopreserved buck spermatozoa.