Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep.

This study was designed to evaluate the relevance of several sperm quality parameters and sperm population structure on the reproductive performance after cervical artificial insemination (AI) in sheep. One hundred and thirty-nine ejaculates from 56 adult rams were collected using an artificial vagina, processed for sperm quality assessment and used to perform 1319 AI. Analyses of sperm motility by computer-assisted sperm analysis (CASA), sperm nuclear morphometry by computer-assisted sperm morphometry analysis (CASMA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed. Clustering procedures using the sperm kinematic and morphometric data resulted in the classification of spermatozoa into three kinematic and three morphometric sperm subpopulations. Logistic regression procedures were used, including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and farm, year, month of AI, female parity, female lambing-treatment interval, ram, AI technician and sperm quality parameters (including sperm subpopulations) as independent factors. Sperm quality variables remaining in the logistic regression model were viability and VCL. Fertility increased for each one-unit increase in viability (by a factor of 1.01) and in VCL (by a factor of 1.02). Multiple linear regression analyses were also performed to analyze the factors possibly influencing ejaculate fertility (N=139). The analysis yielded a significant (P<0.05) relationship between sperm viability and ejaculate fertility. The discriminant ability of the different semen variables to predict field fertility was analyzed using receiver operating characteristic (ROC) curve analysis. Sperm viability and VCL showed significant, albeit limited, predictive capacity on field fertility (0.57 and 0.54 Area Under Curve, respectively). The distribution of spermatozoa in the different subpopulations was not related to fertility.

Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype.

We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus-like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18-20 cm; normal range, 15-16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21-24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated.

In vitro assessment of sperm quality from rams of high and low field fertility.

The aim of the present study was to investigate whether differences in field fertility of rams are reflected in differences in several sperm quality parameters. Ejaculates from 8 adult rams, 4 with high and 4 with low field fertility, were collected weekly using an artificial vagina over 6 consecutive weeks. Analyses of sperm motility by computer-assisted sperm analysis (CASA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed at 0, 3, 6 and 24h of incubation at 37°C. Sperm nuclear morphometry was also determined at 0h by computer-assisted sperm morphometry-fluorescence (CASMA-F). Sperm viability and most CASA sperm motility parameters were higher at 0, 3 and 6h in the high fertility rams. These rams had also a higher sperm nuclear area, perimeter and length (P<0.05) determined by CASMA-F. Significant differences between high and low fertility groups were also found in the dynamics in DNA fragmentation, with significant differences at 6h (14.42±1.40 and 20.27±1.77, respectively, P<0.05) and at 24h (22.32±2.03 and 31.24±2.54, respectively, P<0.01). It was concluded that high and low fertility rams present clear differences in several sperm quality parameters. This opens up the possibility of selection of males for artificial insemination based on sperm quality data.

Climate factors affecting fertility after cervical insemination during the first months of the breeding season in Rasa Aragonesa ewes.

This study was carried out to examine the impact of several climate variables on the pregnancy rate after cervical artificial insemination (AI) of Rasa Aragonesa ewes. Data were derived from 8,977 inseminations in 76 well-managed flocks performed during the first month of the breeding season (July to October). The following data were recorded for each animal: farm, year, month of AI, parity, lambing-treatment interval, inseminating ram, AI technician, and climatic variables such as mean, maximum and minimum temperature, mean and maximum relative humidity, rainfall, and mean and maximum temperature-humidity index (THI) for each day from day 12 before AI to day 14 post-AI. Means were furthermore calculated for the following periods around AI (day 0): -12 to 0, -2 to 0, AI day, 0 to 2, and 0 to 14. Logistic regression analysis indicated that the likelihood of pregnancy decreased when maximum temperature in the 2 days prior to AI was higher than 30 °C (by a factor of 0.81). Fertility was also lower for primiparous ewes and in multiparous ewes with more than five previous parturitions. Other factors with significant impact on fertility were flock, technician, inseminating ram, and a lambing-AI interval longer than 240 days. It was concluded that the 2 days prior to AI seems to be the period when heat stress had the greatest impact on pregnancy rate in Rasa Aragonesa ewes.

Factors affecting fertility after cervical insemination with cooled semen in meat sheep.

Field results of 18,328 cervical artificial inseminations (AI) with cooled semen in Rasa Aragonesa meat sheep under field conditions in north-eastern Spain AI were analyzed. Logistic regression procedures were used including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and year, month of AI, farm, hours between extraction and insemination, number of ewes inseminated in a set of AI, parity, lambing-treatment interval, total number of synchronization treatment per ewe, inseminating ram and AI technician as independent factors. Previous parturitions, lambing-AI interval, month, farm, inseminating ram and technician were factors with significant impact on AI fertility. Based on the odds ratio, the likelihood of pregnancy decreased: in ewes with more than five previous parturitions (by a factor of 0.87, 0.79 and 0.66 for the 6th, 7th and ≥8 parturitions, respectively); in ewes with lambing-AI interval higher than 240 days (by a factor of 0.8); and for inseminations performed during the spring period, (March, April, May and June, 0.70, 0.76, 0.66, and 0.76, respectively). We noted a higher fertility in seven inseminating rams (odds ratios between 1.4 and 1.7) and lower in two rams (odds ratios between 0.6 and 0.7). Of the 17 AI technicians, two were related to fertilities improved by odds ratio of 1.6, and 1.30, whereas two technicians were attributed fertility rates reduced by odds ratios of 0.68 and 0.40. These findings should be taken into account to evaluate the AI technique performance and make decisions to enhance fertility results.

Effect of the FecX(R) polymorphism in the bone morphogenetic protein 15 gene on natural or equine chorionic gonadotropin-induced ovulation rate and litter size in Rasa Aragonesa ewes and implications for on-farm application.

A new mutation in the bone morphogenetic protein 15 (BMP15) gene (FecX(R) allele) causing increased prolificacy in heterozygous (R+) and sterility in homozygous ewes has been recently described in Rasa Aragonesa, a low-prolificacy Mediterranean breed. The current study determined, first, the effect of this polymorphism on natural and eCG-induced ovulation rate (OR) and the effect of eCG dose on reproductive performance; and second, its effect on prolificacy and its interaction with progestagen + eCG treatment on farms, which have not been reported to date. The FecX(R) allele increased OR by 0.44 and 0.63 ovulations in young (n = 91) and adult (n = 84) R+ ewes, respectively (both, P < 0.01), increments less than reported in prolific breeds carrying other mutations in BMP15. When the standard dose of eCG used on farms (480 IU) was applied to R+ ewes (n = 36), an extremely high OR (3.95) was recorded, which was accompanied by greater partial failure of multiple ovulations (PFMO). On the contrary, OR using 240 IU in R+ ewes (2.90; n = 35) was similar to 480 IU in wildtype (++) ewes (2.82; n = 48; both P < 0.01 when compared with 480 IU in R+ ewes). No differences were found in the birth weight of the offspring between R+ and ++ eCG-stimulated ewes within the same litter size. To validate the genealogy identification on farms, PCR genotyping was carried out in 1,667 ewes from 4 elite flocks, resulting in a negligible misclassification of R+ ewes, which demonstrated that identification by genealogy is a reliable tool to identify FecX(R) ewes within the breeding program. In recorded farms, the natural litter size of ++ ewes (1.34, n = 599,160 lambing records) was increased due to the FecX(R) allele by 0.35 lambs (P < 0.0001, n = 6,593 lambing records). A similar increase (0.30) was observed when comparing ++ and R+ ewes treated with 480 IU of eCG (P < 0.0001, n = 62,055 and n = 866, respectively). When applying 480 IU of eCG to R+ ewes, the increase in prolificacy was only due to increased percentages of triplets (P < 0.001) and quadruplets (P < 0.0001), but not of twin births. In conclusion, the favorable reproductive performance of R+ ewes, with 0.63 extra ovulations and 0.35 extra lambs per lambing ewe, is responsible for the increased interest in the use of this polymorphism. Nevertheless, care must be taken in the application of eCG to R+ ewes, with the current results showing that the standard dose increases prolificacy by only increasing triple and higher-order births.

Novel group A rotavirus G8 P[1] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs.

Rotavirus is a worldwide major cause of diarrhea outbreaks in neonatal ruminants. An outbreak of ovine diarrheic syndrome (ODS) in 50-75 days-old lambs (weaned lambs) is described. Fecal immunochromatography and intestinal immunohistochemistry for rotavirus group A were performed. In addition, semi-nested multiplex RT-PCR for G and P rotavirus genotyping in combination with sequencing were performed, to support the diagnosis and identify the viral strain. A novel ovine rotavirus group A G8 P[1] strain was determined as the main cause of the ODS observed, whereas other pathogens were ruled out.

A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep.

Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta superfamily, is specifically expressed in oocytes and is essential for sheep prolificacy. Reported mutations in this gene cause increased ovulation rate and infertility in a dosage-sensitive manner. In this work, a new naturally occurring mutation in the BMP15 gene from the ovine Rasa Aragonesa breed is described. This mutation is a deletion of 17 bp that leads to an altered amino acid sequence and introduces a premature stop codon in the protein. Highly significant associations (P < 0.0001) were found between the estimated breeding value for prolificacy and the genotype of BMP15 in Rasa Aragonesa animals with high and low breeding values for this trait. As for other mutations in BMP15, this new mutation is associated with increased prolificacy and sterility in heterozygous and homozygous ewes respectively.

Prediction of fertility by centrifugal countercurrent distribution (CCCD) analysis: correlation between viability and heterogeneity of ram semen and field fertility.

The prediction of the fertilizing ability of a sire or a given insemination dose is a primary aim in the field of artificial insemination. Centrifugal countercurrent distribution analysis (CCCD) was used to determine the relationship between some sperm parameters and the in vivo fertility rate obtained with the same sample after cervical artificial insemination. A total of 522 ewes from 26 different farms was inseminated with 53 ejaculates obtained from 25 mature Rasa aragonesa rams. Semen was diluted to 1.6 x 10(9) cells ml-1 and doses of 0.25 ml were prepared and kept at 15 degrees C until used for insemination. The same ejaculates were used for analysis of standard semen parameters and CCCD analysis. Sperm motility, concentration and viability were determined before and after CCCD. Post-CCCD parameters were derived from the analysis of the profile obtained after CCCD. The recovered viability showed the highest correlation with fertility, especially in the central chambers (V2), r = 0.415, P < 0.005). The ejaculate heterogeneity also showed a positive correlation with field fertility (r = 0.23), with a tendency towards significance (P < 0.1). The mean fertility value of all ejaculates used in this study was 46.75%, ranging from 12.5% to 75.0%. Ejaculates were classified into two categories according to their fertility: higher and lower than the mean value. Only the viability recovered in the central chambers (V2) was a parameter with a predictive capacity to discriminate between the two groups (P < 0.05). A predictive equation for field fertility with a correlation coefficient r = 0.488 and a very high level of significance (P < 0.005) was deduced by multiple analysis: PF = 6.02 + 0.069V2 + 0.315H (where PF is predictive fertility, V2 is the recovered viability in the CCCD profile central chambers and H is heterogeneity).