Relation between stallion sperm binding to homologous hemizonae and fertility

The hemizona assay (HZA) has been developed as a diagnostic test to predict the fertilisation potential of human spermatozoa. The aim of this study was to develop an HZA for stallion spermatozoa and to investigate a possible relationship between fertility and the outcome of the HZA in this species. Equine oocytes were obtained from ovaries collected at a slaughterhouse and by transvaginal, ultrasound-guided follicle aspiration. They were then denuded from cumulus cells and stored in salt solution at 4 degrees C until use. On the day of the experiments the oocytes were bisected, thus providing 2 equal matching hemizonae from each oocyte. Semen samples from Dutch Warmblood stallions with known fertility data were used to assess the number of spermatozoa bound to the outer side of the hemizona after incubation in vitro. Sperm binding to matching hemizonae of a particular stallion was similar and confirmed the feasibility of using the HZA for the horse. Sperm hemizona binding capacity of 10 pairs of stallions was compared by incubating 1 hemizona with the semen of a stallion and the matching hemizona with the semen of another stallion from the same stud farm. Five matching pairs of hemizonae were used for each pair of stallions. There was a significant relationship between the mean number of spermatozoa bound to matching hemizonae and the fertility indices of stallions from each stud farm (P < 0.0001). It is concluded that HZA can be used as a valuable parameter in stallion semen analysis.     

Catecholamines in fetal pig plasma and the response to acute hypoxia and chronic fetal decapitation

Summary Dopamine, norepinephrine and epinephrine were measured by radioenzymatic assay in blood plasma samples drawn from the umbilical arteries of 30 anaesthetised Landrace pig fetuses. Just prior to term, the concentrations of dopamine (0.46±0.14 ng·ml^–1) and norepinephrine (1.74±0.60 ng·mg^–1) were lower than earlier in gestation, whereas epinephrine concentrations at term (0.80±0.31 ng·ml^–1) were similar to those at mid-gestation, intervening stages of gestation having higher levels of plasma epinephrine. Fetal hypoxia was induced by clamping the umbilical cord for 2 min and the catecholamines determined in arterial blood samples immediately thereafter, then again 3 min after removal of the clamp. Inconsistent effects of cord clamping on catecholamine levels were seen at 55 days, but thereafter, in all but one instance, the hormone levels were increased. Fetuses near term tended to respond less than fetuses at 75 and 96 days gestation (term=114±1 day). Catecholamines were also present in the circulation of fetuses decapitated at 42 days gestation and studied at 109±1 days. The average concentrations of dopamine (1.12±0.27 ng·ml^–1) and norepinephrine (8.23±3.04 ng·ml^–1) were greater than in intact fetuses, the plasma epinephrine levels being comparable to, or slightly higher than, those in intact fetuses. The results demonstrate that catecholamines are present in the circulation of the intact and decapitated pig fetus and that the actual concentrations and the type of response to umbilical cord clamping are dependent on gestation age.     

Changes in serum LH concentrations during normal and abnormal sexual development in the pig.

Serum levels of luteinizing hormone (LH) were determined in fetal and prepubertal pigs from Day 49 postcoitum to 25 weeks of age, and also in unilaterally cryptorchid, freemartin and castrated pigs of similar ages. Serum LH was undetectable in the second trimester of pregnancy, but then gradually increased up to 2 weeks after birth in both sexes. 2-week-old pigs showed irregular LH peaks exceeding 2 ng/ml. Serum LH concentrations decreased during the 3rd and 4th weeks of life. Mean LH concentrations were approximately 2 ng/ml in castrated pigs and 1.3 + or -.7 ng/ml in freemartins. The differentiation and functional activity of the testis appeared to be well correlated with the changes in serum LH concentrations. Changes in serum LH could not be correlated with normal and abnormal gubernacular development.     

Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma.

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.     

Arylsulfatases are present in seminal plasma of several domestic mammals.

Mammalian spermatozoa and seminal plasma both contain high levels of arylsulfatases (AS), enzymes that remove sulfate from sulfated glycoconjugates. In ejaculated semen of boars, 85% of AS was found in seminal plasma whereas only 13% was found in spermatozoa. A comparable distribution of AS between spermatozoa and seminal plasma was observed in other domestic mammals. The presence of AS in seminal plasma was not due to leakage from spermatozoa because sperm cells had intact acrosomes and plasma membranes after their separation from seminal plasma, and because 84% of the acrosomal marker enzyme hyaluronidase was retained in washed spermatozoa. Spermatozoa in boar semen diluted with Beltsville Thawing Solution (BTS) deteriorated faster during storage at 17 degrees C than spermatozoa stored in BTS without seminal plasma. This suggests that seminal plasma has a deleterious effect on mammalian spermatozoa. We propose that (1) sulfated glycoconjugates stabilize sperm plasma membranes; (2) AS present in seminal plasma contribute to the deterioration of spermatozoa by desulfating these glycoconjugates; and (3) AS present in seminal plasma could well play a role in sperm capacitation.     

Variables associated with peripartum traits in dairy cows. III. Effect of diets and disorders on certain blood traits

BLood samples were collected from 89 Holstein cows on days 220 and 250 of gestation, within 24 hr prepartum and postpartum and on day 30 postpartum. Balanced diets which contained either chopped hay (29 cows), hay crop silage (HCS; 30 cows) or corn silage (CS; 30 cows) were fed from day 220 of gestation to day 30 postpartum. The purpose was to determine if variations in certain blood traits were indicative of peripartum and postpartum disorders. The blood traits evaluated were concentrations of plasma total protein, whole blood hemoglobin, packed cell volume, and white blood cells, and serum glutamic oxalacetic transaminase (SGOT), glucose, urea nitrogen, calcium, inorganic phosphorus, magnesium, potassium and sodium. No blood trait was useful to predict a disorder prior to its visual signs with one possible exception. Serum glucose and calcium were lower and SGOT and magnesium were higher peripartum which was prior to death of three cows from fat cow syndrome.     

Isolation and characterization of boar spermatozoa with and without a cytoplasmic droplet

1. Boar semen was separated on a Percoll density gradient into three populations; a low-density band of immature sperm cells containing a cytoplasmic droplet and a high-density doublet band formed by spermatozoa without a cytoplasmic droplet. 2. In these three cell populations four acid hydrolases were determined, viz. (1) alpha-glucosidase; (2) alpha-mannosidase; (3) beta-galactosidase; (4) beta-hexosaminidase. 3. The release of the hydrolases (1), (2) and (3) from cytoplasmic droplet containing spermatozoa was stimulated whereas the release of beta-hexosaminidase was inhibited by calcium ions. 4. The results suggest that acid alpha-glucosidase, alpha-mannosidase and beta-galactosidase are situated in the acrosome whereas acid beta-hexosaminidase is localized predominantly in the cytoplasmic droplet of boar spermatozoa. 5. We conclude that beta-hexosaminidase should prove useful as a biochemical marker for cytoplasmic droplet containing spermatozoa and hence for the number of immature sperm cells in boar semen.     

The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer

This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen (n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5+/-0.4 ml, 77+/-6% and 1471.3+/-940.0 x 10(6) ml(-1), respectively. Post-thaw motility in respective extender was A: 66+/-16%; B: 71+/-2%; C: 73+/-6%; D: 9+/-4% and E: 26+/-12% (mean+/-S.D.). In extender C (74+/-14%) more viable spermatozoa were preserved than in the others (A: 64+/-10%; B: 48+/-11%; D: 41+/-16%; E: 47+/-6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used (P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3+/-0.5 ml, 82+/-4% and 379.1+/-252.2 x 10(6) ml(-1), respectively. Post-thaw motility was in respective extenders A: 69+/-2%; B: 74+/-6%; C: 73+/-2%; D: 13+/-6% and E: 31+/-20%. Extenders B and C were superior (P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70+/-7%), B (76+/-7%) and C (79+/-2%) was higher than that D (25+/-19%) and E (29+/-17%) (P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86+/-4%) and C (83+/-4%) than in extenders A (54+/-13%), D (39+/-22%) and E (46+/-22%) (P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation (P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk-Tris-Tes-glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk-Tris-citric acid-glycerol based extender B containing Equex and extender C are superior in semen cryopreservation to others for Formosan Sambar deer.     

Effects of in vitro production on horse embryo morphology,cytoskeletal characteristics,and blastocyst capsule formation

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.     

Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate

The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a final concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF significantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus-oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone.