The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.     

An In Vitro Evaluation of Biochemical Processes Involved in Lead‐Induced Changes on Ram Spermatozoa

Lead (Pb²⁺) is a toxic heavy metal which interferes with several physiological processes regulated by Ca²⁺, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb²⁺/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb²⁺ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb²⁺ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb²⁺ action. On the contrary, Pb²⁺‐incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb²⁺ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb²⁺ in spermatozoa; thus, the enhancement of Pb²⁺ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb²⁺ by spermatozoa. However, the increase of intracellular Pb²⁺ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.     

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll^®, Puresperm^® and Bovipure^?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure^? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll^® and Puresperm^® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll^® selected less live and more apoptotic spermatozoa than Puresperm^® and Bovipure^? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

A Study on Postpartum Metritis in Iraqi Buffalo Cows: Bacterial Causes and Treatment

The objectives of the present study were to determine the relationship between bacteriological findings, clinical signs and histopathological changes in postpartum metritis. Evaluation of the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics with some hormonal preparations for the treatment of postpartum metritis. Data were collected from 50 buffalo cows with history of calving of more than 1 month. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from uterine lumen from each buffalo included in the present study. Bacteria identified using API systems following aerobic and anaerobic cultures. Vaginal mucus scored for character, odour and estimation of polymorphonuclear cells (PMNs). Treatment conducted using oxytetracycline in local intrauterine infusion or systemically with hormonal treatment including prostaglandinF2α (PGF2α) and oestradiol benzoate. Results revealed that the most predisposing factor for postpartum uterine infection was retained placenta and toxic puerperal metritis. The most prevalent bacteria in uterine lumen were Escherichia coli, Archanobacterium pyogenes, Bacteroides fragilis and Fusobacterium necrophorum the most prevalent bacteria in buffaloes with postpartum metritis. A. pyogenes and F. necrophorum were an important pathogens causing severe uterine inflammation as found in histopathological examinations. Buffaloes with postpartum metritis showed good clinical cure when oxytetracycline injected systemically with PGF2α. Intrauterine infusion of oxytetracycline had no advantage for the treatment of uterine infection in buffalo cows with postpartum metritis. PGF2α improved clinical cure of buffaloes with postpartum metritis.