Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm

The objective was to evaluate the effects of timing of insemination and type of semen in cattle subjected to timed artificial insemination (TAI). In Experiment 1, 420 cyclic Jersey heifers were bred at either 54 or 60 h after P4-device removal, using either sex-sorted (2.1 × 10⁶ sperm/straw) or non-sorted sperm (20 × 10⁶ sperm/straw) from three sires (2 × 2 factorial design). There was an interaction (P = 0.06) between time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88). In Experiment 2, 389 suckled Bos indicus beef cows were enrolled in the same treatment groups used in Experiment 1. Sex-sorted sperm resulted in lower P/AI (41.8%; 82/196; P = 0.05) than non-sorted sperm (51.8%; 100/193). In addition, there was a tendency for greater P/AI (P = 0.11) when TAI was performed 60 h (50.8%; 99/195) versus 54 h (42.8%; 83/194) after removing the progestin implant. In Experiment 3, 339 suckled B. indicus cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occured 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%) or >24 h (5.8%) before ovulation. In conclusion, sex-sorted sperm resulted in a lesser P/AI than non-sorted sperm following TAI. However, improvements in P/AI with delayed time of AI were possible (Experiments 1 and 3), and seemed achievable when breeding at 60 h following progestin implant removal, compared to the standard 54 h normally used in TAI protocols.     

Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors

Two experiments were designed to evaluate the effect of different insemination times (12 and 24h or 18 and 30h) and different types of semen (sex-sorted or non-sorted sperm) on embryo production in Nelore (Bos indicus) and Holstein (Bos taurus) superstimulated donors. In the first experiment, hormonal superstimulation of ovarian follicular development in Nelore donors (n=71) was performed in randomly allocated animals to one of the three treatment groups, and they were inseminated at 12 and 24h after an ovulatory stimulus with pLH treatment was applied, either with sex-sorted (4.2×10(6) sperm/insemination; S12/24; n=17) or non-sorted sperm (20×10(6) sperm/insemination; NS12/24; n=18), or they were inseminated at 18 and 30h using sex-sorted sperm (4.2×10(6) sperm/insemination; S18/30; n=19). A greater number of transferable embryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30h (4.5±3.0) compared to insemination at 12 and 24h (2.4±1.8; P<0.001). However, a greater embryo production (6.8±2.6) was obtained with non-sorted sperm. In the second experiment, the same insemination times and semen types were used in lactating high-production Holstein cows (n=12). A crossover design was employed in this trial. A lesser embryo production (P=0.007) was found in Holstein donors that were inseminated using sex-sorted sperm at 12 and 24h (4.6±3.0) compared to non-sorted sperm (8.7±2.8). However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30h (6.4±3.1). These results supported the current hypothesis that it is possible to improve embryo production using sex-sorted sperm in B. indicus and B. taurus superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for timed artificial insemination (TAI) with sex-sorted sperm was still less than the production with non-sorted sperm.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm

Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting.     

The effect of sperm and cryoprotectant concentration on the freezing success of sex sorted ram sperm for in vitro fertilization

The current method used to sex-sort ram sperm resulted in a dilute end product. The obligatory removal of cryopreservation medium during preparation of sperm IVF further reduced sperm number. This study aimed to increase the number of viable, sex-sorted sperm available for IVF by increasing their pre-freeze concentration and assessing the cryodiluent concentration used to accommodate this change. In Experiment 1, semen was collected from Merino rams (n = 3), sex-sorted, and then frozen at concentrations of 20, 40, or 80 × 10⁶ sperm/mL in three forms of tris-citrate-glucose cryodiluent containing 5% (L-Cryo), 6% (M-Cryo), and 8% (H-Cryo) (v/v) glycerol. Motility, plasma membrane and acrosome integrity, and mitochondrial activity were assessed at 0, 2, 4, and 6 h post thaw. In Experiment 2, cleavage and blastocyst development rates were compared between non-sorted and sex-sorted sperm frozen at the aforementioned concentrations (in the cryodiluent most effective in Experiment 1). In Experiment 1, total motility between 0 and 6 h was similar for all sperm concentrations when frozen using L-Cryo. Mitochondrial activity was elevated in samples frozen in L-Cryo and M-Cryo at 0 h compared to those preserved in H-Cryo for all concentrations (P < 0.05). In Experiment 2, sex-sorted sperm with a higher pre-freeze concentration yielded a higher sperm concentration after preparation for IVF (8.57 ± 1.22 sperm/mL), compared to the lowest group (2.96 ± 0.18 sperm/mL; P < 0.05). There were no significant differences between non-sorted and sex-sorted sperm for rates of embryo cleavage or development. Therefore, sex-sorted sperm was effectively cryopreserved at a higher concentration than conventionally practiced. Although this yielded a higher sperm concentration for IVF, reduced insemination volume, and increased the number of potentially fertile gametes from which to select, fertilisation rate was not significantly improved.     

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.     

Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction

Previous studies indicate that sex-sorted sperm exhibit different physiology, including fertilizing capacity, from non-sorted sperm. However, differences between X- and Y-bearing sperm in their ability to undergo an acrosome reaction have never been investigated. This study determined the ability of non-sorted and sex-sorted sperm to undergo the acrosome reaction prior to and after cryopreservation. Sperm were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction and the percentages of live-acrosome-reacted sperm and dead sperm were evaluated. The X- and Y-bearing sperm reacted similarly to the PC12 treatment, regardless of whether sperm were assessed prior to or after cryopreservation. Fresh control sperm exhibited lower percentages of live sperm (60%) than either X- or Y- sorted sperm (69-74%, P<0.05). Percentages of live control sperm were also lower after thawing (29-35%) than sex-sorted sperm (55-58%, P<0.05). Control and sex-sorted fresh sperm responded similarly to PC12 treatment. However, sex-sorted cryopreserved sperm exhibited higher percentages of live-acrosome-reacted sperm (23%) than control sperm (9%, P<0.05) after 40 min without PC12 treatment. In addition, cryopreserved control sperm treated with 79 microM PC12 exhibited higher percentages of live-acrosome-reacted sperm than sex-sorted sperm. In conclusion, X- and Y-bearing sperm responded similarly to PC12 treatment. In addition, fresh sexed and non-sorted sperm responded similarly to PC12 treatment. However, cryopreserved sex-sorted sperm underwent an acrosome reaction more rapidly in the absence of PC12 (over a 40 min period) than the non-sorted sperm. Therefore, sex-sorting induced changes in sperm membranes that accelerated the acrosome reaction process in sperm after cryopreservation.     

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.     

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.     

Functional integrity of sex-sorted, frozen-thawed boar sperm and its potential for artificial insemination

Despite being developed in its present form 20 years ago, sex-sorting of mammalian sperm is still a work in progress. While relatively successful in cattle and sheep, the unique challenges of incorporating sex-sorted sperm into pig production have not yet been overcome. Generally speaking, boar sperm survive freeze-thawing less well and are required in larger numbers for insemination, while in vitro embryo production of pig embryos is less successful compared to other domestic species [Niemann H, Rath D. Progress in reproductive biotechnology in swine. Theriogenology 2001;56:1291-1304]. Due to the large number of sperm required for artificial insemination in pigs, a technique of storing sperm after sorting must be developed while adequate numbers of sperm are allocated into X- or Y-chromosome-bearing enriched pools. Cryopreservation is perhaps the ideal method of storage between sorting and insemination, as it allows unlimited time to build up a sperm bank, whereas liquid-storage requires the use of sperm within days of sorting. The limited number of studies investigating the survivability of sex-sorted, frozen-thawed boar sperm have produced promising in vitro results but poor in vivo outcomes. Before fertility can be improved, the causes of any damage to sperm function during the sex-sorting and freeze-thawing procedures must be more fully understood. Once defined, the source of damage may be minimised and this would lead to increased success rates after in vivo application of sex-sorted, frozen-thawed boar sperm.     

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.     

Birth of offspring of pre-determined sex after artificial insemination of frozen-thawed, sex-sorted and re-frozen-thawed ram spermatozoa

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 x 10(6) motile non-sorted, frozen-thawed (Control50), 15 x 10(6) motile non-sorted, frozen-thawed (Control15), 15 x 10(6) motile sex-sorted, frozen-thawed (SF15) or 15 x 10(6) motile frozen-thawed, sex-sorted, re-frozen-thawed (FSF15) ram spermatozoa. Separation of SF15 and FSF15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control15 (36/74; 48.6%), SF15 (35/76; 46.1%) and FSF15 (26/72; 36.1%) groups (P>0.05). A higher percentage of ewes produced lambs in the Control50 (38/70; 54.3%) than the FSF15 group (P<0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen-thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen-thawed, sex-sorted, re-frozen-thawed spermatozoa. This study demonstrated for the first time in any species that frozen-thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.     

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.     

Immunization against inhibin enhances both embryo quantity and quality in Holstein heifers after superovulation and insemination with sex-sorted semen

The objective was to investigate the feasibility of improving embryo yield in superovulated cows following insemination with sex-sorted semen by prior immunization against inhibin. Twenty-eight heifers were allocated into three groups: High (n = 10), Low (n = 10), and Control (n = 8). The High group received one primary (1 mg) and two booster (0.5 mg) vaccinations (28-d intervals) with a recombinant inhibin α-subunit in 1 mL of white oil adjuvant, whereas the Low group received half that dose, and the Control group received only adjuvant. After the last immunization, all heifers underwent a standard superovulation treatment (decreasing doses of pFSH for 4 d), followed by two AI with 2 × 10⁶ sex-sorted semen after the onset of estrus. Inhibin-immunized heifers had higher (P < 0.01) plasma antibody titres, and an earlier onset of estrus (P < 0.05) than Control heifers. The total number of embryo/ova, transferable, and grade 1 embryos in the High group (15.4 ± 1.9, 5.7 ± 0.7, and 3.8 ± 1.0, respectively) was significantly greater than that of the Control group (9.1 ± 1.2, 3.1 ± 0.5, and 0.6 ± 0.2), but was intermediate (P > 0.05) in the Low group (13.0 ± 2.3, 4.4 ± 0.7, and 1.2 ± 0.3). There were no significant differences among groups in number of unfertilized ova and degenerated embryos. The High group also had higher (P > 0.05) plasma progesterone concentrations on the day of embryo collection. In conclusion, immunization against inhibin improved both embryo quantity and quality following superovulation and insemination with sex-sorted semen.     

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.