Marianne Vidament

Centrifugation and addition of glycerol at 22 °C instead of 4 °C improve post-thaw motility and fertility of stallion spermatozoa

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.

Fertility prediction in stallions

Abstract The prediction of a stallions fertility is one of the main unresolved problems in horse breeding. Despite many studies of the relationship between seminal characteristics and fertility, we are not able to evaluate with certainty the breeding capacity of a stallion. Stallion selection and fertility prediction are routinely based on the analysis of seminal and behavioural parameters. Basal blood hormone concentrations and their modification after different stimulation are also used for diagnosis. Although the methods developed in some laboratories may explain established subfertility or infertility, mainly detected by abnormal seminal characteristics and/or pregnancy rate, they cannot yet be used for general infertility detection. Such tests are based on spermatozoal morphology, e.g. chromatin and membrane integrity analyses, and spermatozoal functionality, e.g. motility evaluation, zona pellucida-sperm binding assay, and on seminal plasma composition. At this stage we have no idea of the frequency of the abnormalities demonstrated by these methods nor any idea of their correlation with subfertility or infertility.

Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h or more in INRA96 extender

This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 °C, 12 °C to 15 °C, respectively). Per cycle pregnancy rate (PC) was measured after one artificial insemination (AI) in uterine body of 200×10(6) total spermatozoa, 7 h (Experiment 1) or 17 h (Experiment 2) before ovulation. In Experiment 1, PC was similar for both conditions (60% (n=40 cycles) vs. 63% (n=40), respectively, 5 stallions×8 cycles). Only velocity VCL and ALH were slightly higher at 15 °C. In Experiment 2, PC was reduced when ambient temperature was low (semen at -0.5 °C to 3 °C; PC=25%) rather than intermediate (semen at 4 °C to 7 °C; PC=53%) or high (semen at 8 °C to 10 °C; PC=50%) (4 stallions×8 cycles) (P=0.002). Sperm stored at -0.5 °C to 3 °C had lower acrosome integrity/responsiveness, similar membrane integrity (HOS test) and motilities, and higher VCL and ALH, than semen stored between 4 and 15 °C. These results demonstrate a wide tolerance of equine sperm to variable positive temperatures and air exposure for 22 h storage and more. However, temperatures close to 0 °C are detrimental for fertility.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.