Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares.

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Factors influencing parentage ratio in canine dual-sired litters.

Breeding a bitch with two different sires during a single estrous cycle has the potential to facilitate rapid genetic gain and improve reproductive performance within a canine breeding colony. There is limited data regarding the factors that contribute to the success of dual-sired litters in domestic dogs, and only anecdotal evidence suggesting that these litters rarely produce offspring from more than one sire. The objective of this prospective clinical study was to investigate multiple factors that likely affect the success of dual-sired litters on whelping rate, litter size and parentage ratio. These factors include: timing of artificial insemination (AI), order of sires, number of AI's per cycle, semen type, sperm quality and age of sire and bitch. Data collected over a 10 year period from twenty-nine estrous cycles (28 individual bitches of 10 different breeds) were evaluated after an initial AI with frozen semen from the 'genetically desired' sire and followed up with a second AI with either fresh (n = 9) or frozen (n = 16) semen or natural mating (n = 4) from a different, 'back up', sire. DNA parentage of each pup born was determined by using a primary panel of 288 SNPs. The whelping rate and litter size from previous single sire inseminations per estrous cycle, in the same bitches, (n = 16) over 25 estrous cycles using either fresh (n = 4) or frozen-thawed (n = 21) semen, were analyzed as controls. Of the 29 dual-sired breedings, 26 bitches whelped (89.7%), and 8 litters (30.8%) were of mixed parentage. In the litters of mixed parentage after a dual-sired breeding, a greater proportion of the offspring were from the second sire than the first sire (73.0% and 27.0% respectively; P < 0.05). Interestingly, in litters where all pups were of single sire parentage after a dual-sired breeding, 50.0% of the offspring were by the first sire and 50.0% were by the second sire. For litters of mixed or single paternity produced by dual-sired breeding there was no difference in average litter size. However, on a per estrous cycle basis for each bitch the whelping rate (89.7% v. 76.0%.) and litter size (5.5 ± 2.5 v. 4.0 ± 2.78) of all dual-sired breedings were greater (P < 0.05) compared to previous single-sired breedings (controls) respectively. This study demonstrates that offspring of mixed parentage derived from dual-sired breedings may be achieved. Furthermore, insemination with semen from two different sires may increase the whelping rate and litter size, which is an important consideration when using genetically valuable, or older individuals with potentially reduced fertility.

Factors affecting the reproductive performance of bitches: A prospective cohort study involving 1203 inseminations with fresh and frozen semen.

The aim of this prospective cohort study was to utilize multivariable statistical methods to identify factors that significantly affected whelping rate, litter size and gestation length in a large population of bitches of many different breeds, presented for routine breeding management. In addition, we aimed to determine the incidence of dystocia and the proportion of bitches undergoing a caesarean section procedure. A total of 1146 individual bitches representing 84 different breeds contributed 1203 inseminations over the 9 year (2007-2015) study period. Bitches were inseminated with either frozen-thawed (n = 645), fresh (n = 543) or chilled (n = 15) semen from 1371 different males. The mean (SD) whelping rate was 74± 4% and the mean litter size was 5.8 ± 3.1 pups per litter for all bitches in the study. The whelping rate was significantly lower in bitches inseminated with frozen-thawed semen compared with bitches inseminated with fresh semen (71% vs 80% respectively; P < 0.001). Semen that was classified as having poor motility (<30% progressive) resulted in a significantly lower whelping rate (37%) than semen classified as good (30-65% progressive; whelping rate = 67%) or excellent (>65% progressive; whelping rate = 79%). There was a linear decline in whelping rate with advancing age. Greyhounds and Labradors demonstrated a significantly higher whelping rate (88% and 94% respectively) compared with all other breeds (71.3%, P < 0.001). Bitches inseminated with frozen-thawed semen had significantly smaller litter sizes than bitches inseminated with fresh semen (5.4 ± 3.1 vs 6.2 ± 3.0 pups per litter respectively; P = 0.02). Smaller breeds had significantly smaller litters (4.4 ± 2.1 pups) than medium (5.2 ± 2.9 pups), large (5.9 ± 2.9 pups) or giant (6.7 ± 3.8 pups) breeds. For each advancing year of age, litter size decreased by 0.13 pups per litter. The mean (SD) gestation length from LH0 was 65 ± 1.9 d. Greyhounds had a significantly longer pregnancy duration (68.0 ± 1.5 d) than other breeds. For each additional year of bitch age, gestation length increased by 0.11 days (P < 0.01), and for each additional pup per litter, gestation length was reduced by 0.08 days (P < 0.05). Of the 890 bitches for which whelping outcomes were recorded; 409 (46%) whelped normally without assistance, 249 (28%) had an elective C-section, 205 (23%) underwent an emergency C-section and 27 (3%) were medically managed or required veterinary assistance for dystocia. Brachycephalic breeds were 11.3 (95CI = 9.3-17.9; P < 0.001) times more likely to have a C-section compared to all other breeds. Bitches with litter sizes of one or two pups had a C-section rate of 83%, whereas bitches with litter sizes of three or more pups had a C-section rate of 43% (P < 0.001). This study provides important clinical information to optimise whelping rates, litter size and the prediction of whelping in certain breeds for clinicians working in canine reproduction.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6 years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1 ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p < .01) lower (1.75-15.6 ng/ml) than small-sized (5.6-24.2 ng/ml), medium-sized (4.3-23.7 ng/ml) and large-sized (4.3-21.0 ng/ml) bitches. The mean AMH concentration in bitches less than 4 years of age was 12.4 ng/ml, whereas the mean AMH concentration in bitches older than 4 years of age was 10.5 ng/ml (p < .05). For each additional year of age above 1 year of age, AMH concentrations fell by 0.5 ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p < .01). For each 1 ng/ml increase in AMH, litter size increased by 0.3 pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

Calcium, parathyroid hormone, oxytocin and pH profiles in the whelping bitch.

Despite the high prevalence of primary uterine inertia in whelping bitches, the underlying pathogenesis remains unclear. The objectives were to i) determine serum concentrations of total calcium, ionized calcium (iCa), parathyroid hormone (PTH), and blood pH in normally whelping bitches throughout the peri-parturient period; and ii) investigate relationships among iCa, PTH, and acid-base status, and the role that they and oxytocin may have in the underlying pathogenesis of canine uterine inertia. Bitches were randomly selected from a population of German Shepherd Dog bitches with a history of uncomplicated parturition (Group 1; n=10), and from a population of Labrador bitches with a clinical history of an increased incidence of uterine inertia and stillbirths (Group 2; n=20). Jugular blood samples were collected daily from -4 d to the onset of whelping (t=0 h), and then every 4h until the last pup was born. Overall, bitches from Group 2 had higher mean+/-SEM serum concentrations of PTH (4.72+/-2.45 pmol/L, P<0.001), lower iCa (1.31+/-0.08 pmol/L, P<0.05), and higher venous pH (7.41+/-0.03, P<0.005) than bitches from Group 1 (2.9+/-1.44 pmol/L, 1.38+/-0.06 mmol/L, and 7.33+/-0.02, respectively) during the periparturient period. However, there was no significant difference between Groups 1 and 2 for serum oxytocin concentrations during the periparturient period (45.5+/-40 and 65.5+/-82 pg/mL). We inferred that low iCa resulting from a rising pH and decreasing PTH during the periparturient period may have contributed to decreased uterine contractility and increased risk of stillbirths. Therefore, manipulating the cationic/anionic difference in diets of pregnant bitches, similar to the bovine model for hypocalcamia, may reduce the incidence of stillbirths in the bitch.

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.

Preservation and artificial insemination of sexed semen in sheep.

Sperm-sexing technology using flow cytometry is in advanced stages of development for the sperm of several species. The sorting process could compromise sperm viability and sperm require specific handling procedures both before and after sorting to maintain the integrity and function of the sorted sperm. Standard freezing protocols have been modified for post-sorting cryopreservation of sperm and frozen sperm have been successfully thawed, sorted, refrozen and subsequently used to produce offspring. The relatively low numbers of available sorted sperm have, in some cases, led to modification of artificial insemination techniques to maximise efficiency of use. Multiple ovulation and embryo transfer, or in vitro fertilisation and associated technology, may lead to the more efficient use of sexed sperm.

Integration of sperm sexing technology into the ART toolbox.

Sex-sorting of mammalian spermatozoa has applications for genetic improvement of farm animals, in humans for the control of sex-linked disease, and in wildlife as a captive management strategy and for the re-population of endangered species. Considerable research has been undertaken worldwide on the Beltsville sperm sexing technology, the only effective method for pre-selection of sex of offspring. The combination of this method with assisted reproductive technologies has resulted in the birth of offspring in a wide range of animals, including cattle, the only livestock species in which sperm sexing is used commercially. Major improvements in the efficiency of sorting, in particular the development of high speed sorting (15 million X and Y spermatozoa per hour) have led to the production of offspring using conventional and low dose AI and the successful cryopreservation of sorted spermatozoa in cattle, sheep, horses and elk. A major limitation remains the short viable lifespan of sorted spermatozoa in the female genital tract, in most species necessitating sperm deposition deep in the uterus, and close to the expected time of ovulation, for acceptable fertility after in vivo insemination. Special deep uterine insemination technology has been employed to produce offspring in pigs and horses using low sperm doses. Considerable attention has been paid to reduction of the damage and capacitation-like changes to spermatozoa that result from flow cytometric sorting and from freezing and thawing. However, high-purity sorting of liquid-stored or frozen-thawed spermatozoa for immediate use, or re-cryopreservation for later use, does not reduce its fertilizing capacity in vitro, allowing its combination with in vitro fertilization or juvenile in vitro embryo transfer to produce blastocysts, and offspring in sheep and cattle after embryo transfer. Further research into sorting and preservation methods that incorporate strategies to prevent destabilization of sperm membranes may improve the fertilizing lifespan of flow cytometrically sorted spermatozoa. With continued improvement in sorting instrumentation and biological handling, sorting efficiency should reach a point where commercially acceptable pregnancy rates may be achieved in a number of species after conventional or deep uterine insemination.

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa.

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.

Liquid storage of flow cytometrically sorted ram spermatozoa.

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen-thawed sex-sorted and re-frozen-thawed ram spermatozoa.

UNLABELLED: The characteristics and functional capacity of ram spermatozoa frozen-thawed prior to and after flow cytometric sorting was assessed after incubation (37 degrees C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (CONTROL); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 +/- 1.5% and Y: 87 +/- 1.1% purity). After 6 h incubation (37 degrees C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 +/- 2.0%) compared with all other treatments ( CONTROL: 36 +/- 3.3%, FSF: 28 +/- 3.1%, FCF: 20 +/- 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 +/- 39.4 vs 31 +/- 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with CONTROL compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and CONTROL spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen-thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.

Flow cytometric sorting of frozen-thawed spermatozoa in sheep and non-human primates.

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen-thawed spermatozoa. The efficacy of sorting frozen-thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85-92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen-thawed samples (FT-NEAT: 32.7 +/- 2.5%; FT-WASH: 32.2 +/- 3.3%; FT-GRADIENT: 73.9 +/- 3.7%) compared with FRESH (83.3 +/- 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 +/- 5.1% and 27.2 +/- 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 +/- 0.9% and 83.3 +/- 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 +/- 2.4% and 71.7 +/- 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 +/- 1.3% and 77.2 +/- 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 +/- 1.7 mm) compared with FT-WASH (29.1 +/- 3.8 mm) and FT-GRADIENT (28.4 +/- 2.0 mm) and similar (P < 0.05) to FRESH (23.7 +/- 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86-97% purity) of frozen-thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 +/- 5.5%; baboon: 37.5 +/- 2.5%; chimpanzee: 73.0 +/- 2.0%), acrosome intact (marmoset: 68.5 +/- 7.5%; baboon: 89.5 +/- 1.5%; chimpanzee: 84.0 +/- 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen-thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.

In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.

Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted X- or Y-chromosome-bearing spermatozoa.

The fertilizing ability of sex-sorted frozen-thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero-tubal junction (UTJ) with a total of 140 x 10(6) unsorted (control) or 2-4 x 10(6) sorted (X or Y) frozen-thawed ram spermatozoa 54 to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo). The number of ewes pregnant after insemination with unsorted frozen-thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.