Frozen-thawed Sex-sorted Sperm Research Articles

Influence of insemination time on the fertility of sex sorted frozen-thawed Y-sperm in red deer

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon® PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01–55:30 h; I_2, 55:31–56:00 h; I_3, 56:01–56:30 h; or, I_4, 56:31–57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples.

Deep insemination with sex-sorted Cashmere goat sperm processed in the presence of antioxidants.

Flow cytometrically sex-sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex-sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex-sorting and freezing process, including ascorbic acid-2-glucoside AA-2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex-sorted fresh and frozen-thawed sperm. Based on these experiments, we performed deep insemination with sex-sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA-2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA-2G supplementation showed an evident protection against the sorting process-induced membrane and acrosome damage. In Experiment 2, AA-2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen-thawed sex-sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA-2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA-2G supplementation efficiently protects quality and function of both fresh and frozen-thawed sex-sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.

Fertility in Dairy Cows After Artificial Insemination Using Sex‐Sorted Sperm or Conventional Semen

The aim of this study was to compare pregnancy per artificial insemination (P/AI) after timed AI with sex-sorted sperm (SS) or conventional semen (CS) in lactating dairy cows. Cyclic cows (n = 302) were synchronized by Ovsynch and randomly assigned into two groups at the time of AI. Cows with a follicle size between 12 and 18 mm and clear vaginal discharge at the time of AI were inseminated with either frozen-thawed SS (n = 148) or CS (n = 154) of the same bull. A shallow uterine insemination was performed into the uterine horn ipsilateral to the side of probable impending ovulation. Pregnancy per AI on Day 31 tended (p = 0.09) to be less for SS (31.8%) than CS (40.9%). Similarly, P/AI on Day 62 was less (p = 0.01) for cows inseminated with SS (25.7%) compared with CS (39.0%). The increased difference in fertility between treatments from Days 31 to 62 was caused by the greater (p = 0.02) pregnancy loss for cows receiving SS (19.2%) than CS (4.8%). Cow parity (p = 0.02) and season (p < 0.01) when AI was performed were additional factors affecting fertility. Primiparous cows had greater P/AI than multiparous cows both on Day 31 (41.7% vs 25.0% in SS and 53.0% vs 31.8% in CS groups) and on Day 62 (33.3% vs 20.5% in SS and 48.5% vs 31.8% in CS groups). During the hot season of the year, P/AI on Day 31 was reduced (p = 0.01) in the SS group (19.6%) when compared with the rates during the cool season (38.1%). In conclusion, sex-sorted sperm produced lower fertility results compared to conventional semen even after using some selection criteria to select most fertile cows.

367 INCIDENCE OF CHROMOSOMAL ABNORMALITIES IN MALE AND FEMALE BOVINE EMBRYOS DERIVED FROM SEX-SORTED SPERM

Previous studies from our laboratories have shown that developmental rates following IVF with frozen-thawed sex-sorted sperm are lower than with non-sorted sperm and that this may be related to a difference in the kinetics of development. In addition, it is known that the incidence of chromosomal abnormalities in in vitro produced bovine blastocysts is significantly greater than that observed in blastocysts derived in vivo (Viuff D et al. 1999 Biol. Reprod. 60 123-128). In this study, FISH with X- and Y-chromosome-specific probes was used to assess the extent of chromosome abnormalities in bovine blastocysts derived from IVF with X-sorted, Y-sorted, or unsorted semen from the same bull (n = 15 blastocysts per group). Individual blastocysts were transferred to a small drop of spreading solution (0.01 N HCl, 0.1% Tween 20) on a glass slide. Following digestion of the zona pellucida and cytoplasm, the isolated nuclei were fixed in 3:1 methanol:glacial acetic acid at 4°C for 24 h. Slides were then air dried, incubated at 60°C overnight and stored at -80°C until FISH analysis. The StarFISH X-Y FISH bovine sex test kit was used following minor modification and optimization of manufacturer protocols for use with blastocyst preparations. Slides were examined with standard fluorescence microscopy. Overall, 80% (36/45) of blastocysts were considered to be mixoploid i.e. were a mixture of normal diploid and aneuploid and/or non-diploid cells (10/15 for X-sorted, 14/15 for Y-sorted, and 12/15 for unsorted; P &gt; 0.05). The severity of the chromosomal aberration was relatively low in all groups, however; in other words, only a small fraction of the total number of cells per embryo were chromosomally abnormal (1.64%, 5.62%, and 7.93% for X-sorted, Y-sorted, and unsorted, respectively). The most common chromosome abnormalities observed were diploid-triploid-tetraploid mixoploidies. Interestingly, 20% (3/15) of blastocysts derived from unsorted sperm were chimeric (XX/XY). If such chimeric embryos had been sexed by PCR they would have been ascribed as males. In addition, FISH with probes for non-sex chromosomes would not have revealed such chimerism. In conclusion, the incidence of chromosomal abnormalities was not different between blastocysts derived from sorted and unsorted sperm. The occurrence of mixed sex chimeras amongst blastocysts derived from unsorted sperm is interesting and requires further study. Whether it represents a previously undetected abnormality of IVF is as yet unknown. MGHwas supported by a Posdoctoral Research Grant (2008-0198) from the Spanish Government.

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.

Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

Two field trials were conducted to determine the fertilising capacity of (i) frozen-thawed, sex-sorted re-frozen-thawed (FSF) dairy bull sperm inseminated close to the time of ovulation, (ii) FSF sperm following large dose insemination, and frozen-thawed, sex-sorted (FS) sperm inseminated within 12 h after sorting. In Trial 1, 24 heifers in synchronised oestrus were observed for standing heat over a 30-min period once every 3 h. Upon observation of standing heat, the size of the pre-ovulatory follicle was tracked by ultrasound every 6 h until ovulation was judged to be imminent. Heifers were inseminated with 4 × 10 6 X-bearing FSF or Control sperm within 6 h of ovulation. Ovaries were scanned 6 h after AI to ensure ovulation had occurred. All 24 heifers displayed standing oestrus and 20 of these subsequently ovulated. The mean length of standing oestrus was 16.8 ± 0.4 h and ovulation occurred 27.6 ± 1.1 h after the onset of standing heat from a pre-ovulatory follicle with a diameter of 16.1 ± 0.3 mm. All 12 heifers that received FSF sperm returned to oestrus < 26 d after AI. Of 8 heifers that received Control sperm, 6 (75%) were confirmed pregnant by ultrasound 7 wk after AI, confirming that the method of AI and herd fertility were sound. In Trial 2 the number of sperm inseminated and the effect of eliminating the post-sort cryopreservation step were investigated. Heifers ( n = 21) were synchronised for oestrus, and inseminated 24 h after the onset of standing oestrus with 10 × 10 6 X-bearing FSF, 4 × 10 6 X-bearing FS, or 10 × 10 6 non-sorted frozen-thawed (Control) sperm. Heifers were observed for return to oestrus from 21 d, and diagnosed for pregnancy 7 wk after AI. Of the 7 heifers that received FSF sperm, one was confirmed pregnant (14.3%) and delivered a female calf. Four heifers inseminated with control sperm became pregnant and calved, but no pregnancies were obtained using FS sperm. The birth of a calf following AI with FSF sperm demonstrates the potential of sorting from frozen-thawed semen, and with further work, may be a promising technique that will give producers access to sexed sperm from a greater range of bulls.

Pregnancy percentage following deposition of sex-sorted sperm at different sites within the uterus in estrus-synchronized heifers

Our objective was to assess the effect on heifer pregnancy rate of deposition at three sites within the uterus of frozen-thawed sex-sorted sperm at a fixed time after estrus synchronization. Estrus was synchronized in 209 heifers by administration of PGF2a 14 days apart. At 80–82 h after the second PGF2a injection, X-chromosomes bearing fractions of semen with 2.2 × 10 6 sperm in insemination dose were used for single insemination into the uterine body (UB-AI, n = 91) or for intracornual deposition in the middle of the uterine horn (MH-AI, n = 57) or close to the utero-tubal junction (UTJ-AI, n = 61). The overall pregnancy rate was 43.1%. Pregnancy rates did not differ ( P > 0.05) among sites of sperm sperm deposition, between the two farms at which the heifers were kept or between the two bulls producing the semen. Within UB-AI, MH-AI and UTJ-AI treatments, pregnancy rates were 41.8%, 49.1% and 39.3%, respectively ( P > 0.05). Pooled across classes for deposition site, pregnancy rate was 25.1% higher ( P < 0.01) for heifers showing strong signs of estrus than for heifers showing weak signs of estrus (45.9 versus 20.8%, respectively). Embryonic and fetal loss from diagnosis of pregnancy to term and at calving equalled 5.6%. Of 88 calves of identified sex, 93.2% were female. In conclusion, pregnancy rates of heifers did not differ significantly following deposition of 2.2 × 10 6 sex-sorted sperm 80–82 h after the second PGF2a injection near the utero-tubal junction, in the middle of the horn or into the uterine body.

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex‐sorted Frozen‐thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.

Ultrarapid refreezing of biopsed mouse embryos

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.