Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

In situ provision of drinking water to grazing dairy cows improves milk production

AIMS: To determine the effect of providing water within the area grazed by dairy cows on milk yield and quality, compared to requiring cows to walk to a distant water trough, on a dairy farm in the Pampa region of Argentina during summer.

METHODS: Holstein dairy cows were allocated to two herds with similar parity, days in milk and milk production. They were grazed in one paddock that was divided in two, with a fixed water trough at one end. Cows were moved twice daily to grazing plots within the paddock. Control cows (n=66) could only access water from the fixed trough, whereas supplemented cows (n=67) also received water from a mobile trough within the grazing plot. Milk production of each cow, and water consumption of the two herds were measured daily over 62 days. Milk composition for each herd was determined weekly from Days 18 to 60 of the study, and grazing behaviour was observed between 08:00 and 16:00 hours on Days 11–15, 19–22 and 39–43.

RESULTS: Over the 62 days of the study, supplemented cows produced 1.39 (SE 0.11) L/cow/day more milk than Control cows (p=0.027). Estimated mean daily water intake was 50.4 (SE 2.1) L/cow/day for supplemented cows and 58.2 (SE 2.7) L/cow/day for Control cows (p=0.004). Percentage total solids in milk was higher for supplemented (12.5 (SE 0.06)%) than Control (12.4 (SE 0.04)%) cows (p=0.047). During the periods of behavioural observation, a higher percentage of cows in the water supplemented than the Control herd were observed in the grazing area (p=0.012).

CONCLUSIONS AND CLINICAL RELEVANCE: This preliminary study demonstrated that provision of water to dairy cows within the grazing plot was beneficial for milk production and composition, and may be associated with longer periods spent within the grazing area, during hot weather in the Pampa region of Argentina.     

Pregnancy‐Induced ISG‐15 and MX‐1 Gene Expression is Detected in the Liver of Holstein–Friesian Heifers During Late Peri‐Implantation Period

The bovine embryonic signal interferon‐τ (IFN‐τ) produced by the trophoblast is known to pass through the uterine fluid towards the endometrium and further into the maternal blood, where IFN‐τ induces specific expression of interferon‐stimulated gene expression (ISG), for example in peripheral leucocytes. In sheep, it was shown experimentally by administration of IFN‐τ that ISG is also detectable in the liver. The objective was to test whether ISG can be detected in liver biopsy specimens from Holstein–Friesian heifers during early pregnancy. Liver biopsies were taken on day 18 from pregnant and non‐pregnant heifers (n = 19), and the interferon‐stimulated protein 15 kDa (ISG‐15) and myxovirus‐resistance protein‐1 (MX‐1) gene expression was detected. The expression of both MX‐1 (p: 24.33 ± 7.40 vs np: 9.00 ± 4.02) and ISG‐15 (p: 43.73 ± 23.22 vs 7.83 ± 3.63) was higher in pregnant compared to non‐pregnant heifers (p < 0.05). In conclusion, pregnancy induced ISG‐15 and MX‐1 gene expression in the liver already at day 18 in cattle.     

Comparison of Synthetic Oviductal Fluid and G1/G2 Medium under Low‐1 Oxygen Atmosphere on Embryo Production and Pregnancy Rates in Nelore (Bos indicus) Cattle

In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low‐oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low‐oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.