J. Rutllant

Factors affecting pregnancy loss from gestation Day 38 to 90 in lactating dairy cows from a single herd.

The present study was designed to establish whether factors such as previous estrus synchronization, corpus luteum and embryo number at the time of pregnancy diagnosis, changes in body condition score, milk production, clinical disease (mastitis or lameness) and the inseminating bull affect pregnancy loss from 38 to 90 days of gestation. We derived data from 601 pregnant lactating dairy cows from a single herd. Pregnancy diagnosis was performed by ultrasonography between Day 38 and 44 following insemination. We also recorded corpus luteum and embryo number at this time. Pregnancy loss was defined as a negative pregnancy diagnosis on the second palpation per rectum undertaken between 90 and 96 days after insemination. Data were analyzed using multiple logistic regression methods. Cows that had an additional corpus luteum were eight times less likely to miscarry. The risk of pregnancy loss was 3.1 times higher in cows bearing twins. A one unit reduction in body condition score from previous partum to 30 days postpartum resulted in a 2.4-fold increase in pregnancy loss. We noted a higher incidence of pregnancy loss in cows inseminated using semen from one of the six bulls used. This particular bull led to a 3.4-fold increase in the rate of pregnancy loss. Logistic regression analysis showed no significant effects of previous estrus synchronization, milk production, clinical disease, body condition at previous partum or at pregnancy diagnosis, or body condition change between previous partum and pregnancy diagnosis. Our findings indicate a positive relationship between the presence of an additional corpus luteum and the maintenance of gestation. Risk factors for pregnancy loss were twin pregnancy, reduced body condition after previous parturition and the inseminating bull.

Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions.

Abstract Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl β-cyclodextrin—which causes cholesterol efflux from the spermatozoa—and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.

Evidence for a functional glycogen metabolism in mature mammalian spermatozoa.

The glycogen content in fresh raw dog spermatozoa was 0.22 ± 0.03 μmol/mg protein. This matched with the presence of a glycogen‐like staining in the head and midpiece. Glycogen levels lowered to 0.05 μmol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 μmol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 ± 0.07, measured in the absence and the presence of glucose 6‐P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 ± 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10–20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol. Reprod. Dev. 56:207–219, 2000.

Effect of reproductive disorders previous to conception on pregnancy attrition in dairy cows

This study was undertaken to determine whether reproductive disorders previous to conception influence pregnancy attrition in dairy cows. Reproductive disorders were registered and pregnancy diagnoses were performed as a part of a reproductive health program at 9 commercial dairy herds in northeastern Spain. Data from 3022 diagnosed pregnant lactating cows were used. Pregnancy diagnosis by palpation per rectum was performed from 33 to 70 d post insemination. Pregnancy attrition was registered when the pregnancy diagnosis was negative at the second palpation carried out between 120 and 150 d following insemination. Data analysis was performed by multiple logistic regression methods. Pregnancy attrition rates were 2.6 and 1.8 times higher in cows with previous pyometra and retained placenta, respectively, than in cows without these disorders. No effect of endometritis, ovarian cysts and repeat breeding was shown on pregnancy attrition. Our results suggest that additional efforts to reduce the risk of retained placenta and pyometra should decrease the incidence of pregnancy attrition in dairy cows.

Rheological and ultrastructural properties of bovine vaginal fluid obtained at oestrus

The properties of cervical–vaginal fluid are under strict hormonal control: and in mammals in which semen is deposited in the anterior vagina, changes produced in these properties can result in a lower or higher resistance to sperm motion. The aim of this study was to determine whether the structural organization of bovine vaginal fluid is related to its rheological properties. Vaginal fluid samples were collected from 41 cows at oestrus: 20 at the middle of oestrus (between 8 and 12 h after starting) and 21 at the end of oestrus (between 18 and 22 h). Flow behaviour was determined using a viscosimeter, and the ultrastructural analysis was performed by scanning electron microscopy. Six samples showed newtonian behaviour: three collected at the middle and three collected at the end of oestrus. Newtonian samples had dense and compact matrices arranged as membranes with rough, irregular surfaces, and sparse, thin filaments (< 150 nm). Non‐newtonian samples collected at the end of oestrus (n = 18) had a higher (P = 0.016) consistency index (K = 944 ± 229 mPa.sn) than those collected at the middle of oestrus (n = 17; K = 237 ± 84 mPa.sn). Thick filaments (> 700 nm) that were either randomly arranged with thinner filaments forming a mesh or heavily cross‐linked by thin filaments (50–150 nm) were observed in all non‐newtonian samples collected at the end of oestrus, while medium‐diameter filaments (between 200 and 500 nm) forming loose networks were observed in non‐newtonian samples collected at the middle of oestrus. These findings indicate a close relationship between the molecular organization of the structural elements of bovine vaginal fluid and its rheological behaviour. Vaginal fluid dramatically reduces its mechanical barrier effect during the course of oestrus but always appears to maintain its three‐dimensional filamentous structure. The images of vaginal fluid showing newtonian behaviour would appear to support previous results, suggesting that this property may be related to bovine infertility.

Posttranslational Processing of PH-20 During Epididymal Sperm Maturation in the Horse

Abstract It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.

Rheological behavior of the vaginal fluid of dairy cows at estrus

Abstract The present study was conducted to evaluate rheological properties of the vaginal fluid in dairy cows. Thixotropy and flow behavior were evaluated. Vaginal fluid samples were collected in 8 cows at the beginning of a natural estrus and then every 8 h for 24 h. Vaginal samples were also collected from 208 cows at the time of insemination within 12 to 24 h interval after the onset of a natural estrus. Time-dependent variations were registered during the estrus. The limit shear stress for flow production (To), the shear stress of equilibrium (Te) and the consistency index (K) decreased at 8 and 16 h and markedly increased 24 h after the beginning of estrus. At the time of insemination, structural variation was registered and the fluids were considered to be thixotropic in samples of 181 cows (87%); in the remaining samples (27 cows), structural variation was not registered and the fluids were considered to be nonthixotropic. For flow behavior values, 178 samples (85.6%) presented non-Newtonian behavior, while 30 behaved as Newtonian fluids. The mean values of the TO and the Te were 6.1 ± 0.69 and 1.9 ± 0.69 pascals, respectively. Consistency index (K) mean was 364.9 ± 41.4 milipascals per secondn Our data show an increase of fluidity at the middle of the estrus period in the cow and a wide variation of rheological measurements of vaginal fluid at the time of insemination.

A scanning electron microscopic study of the peritoneal mesothelium covering the genital tract and its ligaments in the cow.

This study was undertaken to describe the surface features of the peritoneal mesothelium covering the genital tract and adjacent ligaments of the cow during the oestrous cycle. The relationship between mesothelial surface and spermatozoa was also evaluated after intra‐uterine and intraperitoneal insemination. Surface features of mesothelial cells from 25 cyclic cows were examined by scanning electron microscopy and by image analysis. Presence of spermatozoa was evaluated by scanning electron microscopy in seven additional cows. In the external side of the infundibulum, the oviductal mucosa exceeds the free margin, forming a continuous band measuring 2.5–10 mm in width. This oviductal epithelium shows cyclical variations with a predominance of ciliated cells during the follicular phase. In respect of the mesothelium, no clear morphological differences were observed associated with the side of ovarian bursa (internal versus external), or with the phase of the oestrous cycle. Mesothelial cells covering the uterus and mesometrium have a higher microvilli density and length and a smaller cell surface area than in the oviduct and adjacent structures. The presence of solitary cilia in the mesosalpinx and mesotubarium superius (infundibulo‐cornual ligament) of some specimens was also observed . When samples were processed without post‐fixation in osmium tetroxide, a layer of amorphous material covered all surfaces. After intra‐uterine insemination of five cows, no spermatozoa were found on their peritoneal mesothelium. Numerous spermatozoa were found after intraperitoneal insemination being attached throughout mesothelial surfaces. These results indicate that there are morphological differences between regions, but no cyclic changes, in the surface features of mesothelial cells covering the genital tract and adjacent ligaments of the cow, and that spermatozoa can bind to mesothelial surfaces after intraperitoneal insemination.

Morphology of a dicephalic cat.

SummaryA detailed anatomical study of a dicephalic iniodymic monosomic cat in conjunction with the morphogenetical implications of the observed anomalies is presented. The animal exhibited two heads joined at the level of an anomalous medial exoccipital bone. Two brains and two foramina magna were present. The vertebral column was single but the cranial cervical vertebrae (C2 to C5) had doubled bodies. Cervical rachischisis with myeloschisis were associated defects. Two nasopharyngeal and oropharyngeal cavities converged caudally into a single laryngopharynx. The esophagus, larynx and trachea were single. Duplication of the tongue and hyoid apparatus was present. Palatoschisis affected both oral cavities. Hypoplasia of the anatomical structures in the medial aspects of both heads was observed. Microphthalmia was also observed in both medial eyes. Comparative aspects of the morphology, causative agents, and mechanisms and anomalous morphogenesis of anterior duplications are reviewed and discussed.