Effects of pH, sodium bicarbonate, cryoprotectants and foetal bovine serum on the cryopreservation of European eel sperm.

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-cryopreservation.     

First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (‘cryolarvae’) were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.     

Measurement of Ovine Pregnancy-Associated Glycoprotein (PAG) During Early Pregnancy in Lacaune Sheep

This study describes ovine pregnancy‐associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi‐purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG_57+59kDa) and R805 (against ovPAG5_58+61kDa) were used respectively in RIA‐780 and RIA‐805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non‐pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA‐780 and 14 900 Ci/mmol in RIA‐805. The minimal detection limits for RIA‐780 and RIA‐805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra‐assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA‐780 and 8%, 9% and 5% for RIA‐805. The inter‐assay CV in the same samples were 13%, 12% and 7% for RIA‐780 and 13%, 11% and 5% for RIA‐805. The recovery was higher than 95% in both assays. No cross‐reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA‐780 and RIA‐805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.     

Paratuberculosis sero-status and milk production,SCC and calving interval in Irish dairy herds

The objective of this study was to investigate the impact of paratuberculosis sero-status on milk yield, fat, protein, somatic cell count and calving interval in Irish dairy herds. Serum from all animals over 12 months of age (n = 2,602) in 34 dairy herds was tested for antibodies to Mycobacterium avium subsp. paratuberculosis using an ELISA. Herds were categorised by sero-status into positive, non-negative and negative, where a positive herd contained two or more positive cows, a non-negative herd contained only one positive cow and a negative herd contained no positive cows. Data at animal, parity and herd-level were analysed by multiple regression using general linear models. Positive herds (mean herd size = 129 cows) and non-negative herds (81 cows) were larger than negative herds (72 cows) (P < 0.01). Negative herds had the highest economic breeding index (EBI), while positive herds had the highest estimated breeding value (EBV) for milk yield. There was no significant effect of paratuberculosis sero-status at animal, parity or herd-level on milk yield, milk fat or protein production, somatic cell count score (SCCS) or calving interval. Negative herds tended to have a lower SCCS than positive and nonnegative herds (P = 0.087). This study only examined the effects of paratuberculosis sero-status but did not examine the clinical effects of Johne's disease at the farm or dairy industry levels.     

64, XX,SRY‐negative,Testicular DSD Syndrome in a Lusitano Horse

Here is reported a disorder of sex development found in the Portuguese Lusitano horse breed. The complex genital phenotype included mammary glands, abdominal testes without epididymis, connected through oviducts to pelvic hypoplastic uterine horns and fused vulvar labia majora from which protruded ventrally a penis‐like structure. This structure was presented in a reversed position, the urethral opening placed dorsally in the glans. However, it was functional both for micturition and erection. The horse exhibited female micturition posture and aggressive male‐like behaviour, including flehmen, mounting, thrusting and flagging of the tail. Plasma testosterone concentrations were below detection limits and the genetic evaluation revealed a 64, XX, SRY‐negative karyotype. Surgery consisted in the removal of abdominal gonads followed by amputation of the penis and repositioning of the urethra. This case of reversion between the chromosomal and gonadal sex, associated with mixed anatomical and behavioural phenotype, illustrates that development of the testes may occur in the absence of the SRY gene and that other genetic and cellular pathways leading to gonad differentiation should be investigated.     

Plasma Concentrations of Pregnancy‐Associated Glycoproteins Measured Using Anti‐Bovine PAG‐2 Antibodies on Day 120 of Gestation Predict Abortion in Dairy Cows Naturally Infected with Neospora caninum

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy‐associated glycoprotein‐2 (PAG‐2) concentrations throughout pregnancy and (ii) whether plasma PAG‐2 concentrations could predict abortion in N. caninum‐infected cows. The study was performed on a commercial Holstein‐Friesian dairy herd in northeastern Spain and the final data included those recorded in 53 non‐aborting and 19 aborting animals. Blood samples were collected immediately before pregnancy diagnosis (on Days 40, 90, 120, 150, 180 and 210 post‐insemination) in non‐aborting cows or until the time of abortion detection in aborting cows. General lineal models (GLM) repeated measures anova revealed the different behaviour of PAG‐1 and PAG‐2, and significant effects of Neospora seropositivity, cool season and twin pregnancy on plasma PAG‐2 concentrations throughout gestation (between‐subject effects). In addition, based on the odds ratios, the likelihood of abortion increased in Neospora‐seropositive cows (by a factor of 7.0) compared to seronegative animals and decreased in cows with a high plasma PAG‐2 concentration (>4.5 ng/ml) on Day 120 of pregnancy (by a factor of 0.24), compared to the remaining cows. In conclusion, there is a relationship between plasma PAG‐2 concentrations and the risk of abortion in Neospora‐infected dairy cows. Thus, plasma PAG concentrations measured using anti‐boPAG‐2 antiserum on Day 120 of gestation could serve as an indicator of the abortion risk in N. caninum infected animals; values <4.5 ng/ml indicating a high risk of abortion in chronically infected animals.