Identification of Sperm-Head Morphometric Subpopulations in Iberian Red Deer Epididymal Sperm Samples

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris–citrate–egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor^®. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser^®, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP₁, SP₂, SP₃) of spermatozoa with different morphometric characteristics (p  <  0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p  >  0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.     

Effect of detomidine or romifidine constant rate infusion on plasma lactate concentration and inhalant requirements during isoflurane anaesthesia in horses

Objective

Influence of detomidine or romifidine constant rate infusion (CRI) on plasma lactate concentration and isoflurane requirements in horses undergoing elective surgery.

The development and integrity of equine pre‐antral follicles cultured in vitro with follicle‐stimulating hormone (FSH) supplementation

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre‐antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre‐antral follicles, the groups with 100 ng/ml FSH of 2‐days culture as well as 50, 100 and 200 ng/ml FSH of 6‐days culture provided the best results. In conclusion, the in vitro culture of abattoir‐derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.     

Determination of the Genital Tubercle Migration Period in Morada Nova Sheep Foetuses by Ultrasonography

The aim of this study was to determine the period of genital tubercle (GT) migration using ultrasonography in Morada Nova sheep foetuses (n = 117) from natural mating (NM) and frozen embryo transfer (ET) to determine the window when foetal sexing can be determined. The examinations were performed using transrectal ultrasonography with a dual-frequency linear transducer (6.0 and 8.0 MHz) from day 30-54 of pregnancy at 48-h intervals. The period of GT migration of foetuses produced by NM varied from 36 to 46 days of pregnancy, resulting in an average of 39.5 +/- 2.9 days. For foetuses derived from ET, GT migration varied from 42 to 52 days of pregnancy with an average of 48.5 +/- 3.3 days, being possible the GT of foetuses from ET start to migrate 96 h later even if they are of the same gender. Migration of the GT occurred earlier (p < 0.05) in foetuses produced by NM and sexing accuracy for triplet pregnancies (77.8%) was significantly inferior (p < 0.05) to single (100%) and twin (92.9%) pregnancies for foetuses derived by NM. The results allow one to conclude that foetal sexing can be done from the 50th day onwards in foetuses produced by NM and from the 55th day onwards in foetuses derived from ET, and that multiple pregnancies compromise the sexing accuracy by ultrasonography.     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

Qualitative and quantitative ultrasound attributes of maternal‐foetal structures in pregnant ewes

The aim of this study was to examine foetal organs and placental tissue to establish a correlation between the changes in the composition of these structures associated with their maturation and the ultrasonographic characteristics of the images. Twenty‐four pregnant ewes were included in the study. Ultrasonography assessments were performed in B‐mode, from the ninth gestational week until parturition. The lungs, liver and kidneys of foetuses and placentomes were located in transverse and longitudinal sections to evaluate the echogenicity (hypoechoic, isoechoic, hyperechoic or mixed) and echotexture (homogeneous and heterogeneous) of the tissues of interest. For quantitative evaluation of the ultrasonographic characteristics, it was performed a computerized image analysis using a commercial software (Image ProPlus^®). Mean numerical pixel values (NPVs), pixel heterogeneity (standard deviation of NPVs) and minimum and maximum pixel values were measured by selecting five circular regions of interest in each assessed tissue. All evaluated tissues presented significant variations in the NPVs, except for the liver. Pulmonary NPVmean, NPVmin and NPVmax decreased gradually through gestational weeks. The renal parameters gradually decreased with the advancement of the gestational weeks until the 17th week and later stabilized. The placentome NPVmean, NPVmin and NPVmax decreased gradually over the course of weeks. The hepatic tissue did not show echogenicity and echotexture variations and presented medium echogenicity and homogeneous echotexture throughout the experimental period. It was concluded that pixels numerical evaluation of maternal‐foetal tissues was applicable and allowed the identification of quantitative ultrasonographic characteristics showing changes in echogenicity related to gestational age.     

Elimination of bovine viral diarrhoea virus in New Zealand: a review of research progress and future directions

The major impacts of bovine viral diarrhoea (BVD) on cattle health and production have prompted many countries to embark on national elimination programmes. These programmes typically involve identifying and removing persistently infected (PI) cattle in infected herds and implementing biosecurity measures, such as pre- or post-movement testing. In order to design a systematic national control programme to eliminate BVD in New Zealand, which achieves the greatest benefits to the industries at the lowest cost to individual farmers, an accurate understanding is necessary of the epidemiology, economics and social motivation for BVD control in New Zealand. In this article we briefly review the pathogenesis of BVD, transmission and diagnosis of BVD virus infection, and effectiveness of vaccination. We summarise the current state of knowledge of the prevalence, risk factors for transmission, and financial impacts of BVD in New Zealand. We describe control programmes in Europe and then discuss the challenges that must be addressed to design a cost-effective national control programme to eliminate BVD in New Zealand.     

Effect of Sildenafil on Pre‐Eclampsia‐Like Mouse Model Induced By L‐Name

N(omega)‐nitro‐L‐arginine methyl ester (L‐NAME) decreases the vasodilator effect of nitric oxide (NO) and induces pre‐eclampsia in mouse. Sildenafil inhibits the degradation of nitric oxide and increases vasodilation. This study aimed to determine the effects of sildenafil citrate on angiogenesis and oxidative stress at the maternal foetal interface on pre‐eclampsia‐like mouse model induced by L‐NAME. Twenty pregnant mice were divided into four groups: (i) vehicle control; (ii) L‐NAME; (iii) sildenafil; (4) L‐NAME+sildenafil. L‐NAME was administered from day 7 of pregnancy and sildenafil from day 8 until day 16; animals were euthanized on day 17. Placental and foetal sizes and weights were measured; lipid peroxide levels and catalase activity in placental homogenates were determined, and placental vascular endothelia were identified by lectin‐histochemistry using BSA‐I lectin. Western blot analysis was used to determine VEGF expression in placental homogenates. No changes were seen in placental and foetal development in mice with normal pregnancies treated with sildenafil. Treatments with L‐NAME reduced significantly the placental weight and average height and decreased the percentage of the endothelial surface. These alterations may be mediated by the reduction of NO levels in trophoblastic cells, due to the inhibitory effect of L‐NAME on nitric oxide synthase (NOS) synthesis. This effect was offset by the treatment with sildenafil, with an increase in the percentage of the endothelial surface. In conclusion, our results indicate that treatment with sildenafil on pre‐eclampsia mouse model can be used without adverse effects on the concept and its use in the treatment of pre‐eclampsia is promising.