Sperm DNA impairment in the bull: causes, influences on reproduction and evaluations

Conventional semen examination involving sperm motility, viability and morphology remains the backbone of assessing the fertility status of a sire. However, there remains instances where these semen parameters appear normal but cases of low conception rates or failure of pregnancy occur. This review highlights the causes of sperm DNA damage and the effectiveness of techniques designed to evaluate the contribution of sperm DNA damage to lowered fertility in bulls. Among the many causes of sperm DNA impairment are imperfect spermatogenesis, faulty apoptosis, reactive oxygen species, in-vitro handling, impact of environment, radiography and the stress of cryopreservation processes. Furthermore, DNA impairment impairs fertilisation, interferes with embryonic development and implantation and blocks blastocyst formation. The most frequently used tests to determine DNA damage are the acridine orange test (AOT) using acridine orange stain with examination under a fluorescence microscope and the sperm chromatin structure assay (SCSA) using the same stain but examined with flow cytometry

Edible birds' nest (EBN) hydrolysate for bovine sperm cryopreservation

The aim of this study was to evaluate the effects of supplementing different concentrations of EBN into Tris (Tr) and Bioxcell (Bx) extenders on bull sperm cryopreservation. A total of 12 semen samples were collected from mature bulls by electro ejaculation. The semen samples were evaluated both freshly and after cryopreservation for quality based on % sperm general and progressive motility (under a microscope), viability and abnormal morphology (using eosin-nigrosin stain). The fresh samples were then diluted and extended using the two extenders containing 0% (control), 0.03%, 0.06%, and 0.12% of EBN. Chilled at 4°C for 3 hours before packaged into 0.25 mL straws and frozen into liquid nitrogen (-196°C) for 48 hours. Results for both extenders revealed insignificant differences (P > 0.05) in all parameters between the different EBN treatment groups and control. Although not significant, 0.12% EBN in both extenders showed the lowest % abnormality, close to the fresh sample reading. In conclusion, EBN concentrations used in this study do not significantly improve sperm quality after freezing. However, the improvement in sperm morphology observed at 0.12% EBN (highest concentration) might imply importance of further increase in dosage for significant effect in future studies

Supplementation of antioxidant BHT to different bull semen extenders enhances semen quality after chilling

The effects of adding various concentrations of antioxidant, butylated hydroxytoluene (BHT) on chilled bull semen for 72 h at 4°C in Bioxcell® (BX), Tris egg-yolk- (TEY) and citrate egg-yolk- (CEY) based semen extenders were studied. Twenty-two ejaculates collected from three bulls, each extended using three extenders with BHT at 0 (control), 0.5, 1.0, 1.5, 2.0 and 3.0 mM/mL, were evaluated for sperm quality parameters. Extended semen was packaged into 0.25 mL straws containing 20 x 106 spermatozoa and chilled for 72 h. Four random straws each from the control and treatment groups were warmed at 37°C, pooled and evaluated using a computer-assisted semen analyser (IVOS Hamilton Thorne Biosciences) for general and progressive motilities, and for morphology, viability and acrosome damage using eosin-nigrosin stain under phase-contrast microscope. Results showed that sperm morphology, viability and protection of acrosome damage were significantly improved (p<0.05) at BHT concentrations of 0.5 in BX and 1.0 mM/mL in TEY and CEY compared to the controls. The BHT also showed a potential positive effect on progressive motility at 0.5 mM/mL in BX and 1.0 mM/mL in TEY and CEY. High concentrations of BHT (2.0 and 3.0 mM/mL), however, produced deteriorative effects on the sperm parameters in all the extenders. In conclusion, BHT when added at 0.5 mM/mL in BX and 1.0 mM/mL concentration to TEY and CEY extenders improved the quality parameters of bull chilled semen

Agreement among bacteriological findings, vaginal discharges, and endometrial cytology for endometritis detection in postpartum beef cows

This study aims to demonstrate the relationship among vaginal discharges, bacteriological contamination, and endometrial cytology during 20–30 days postpartum in beef cows. A total of 82 beef cows, aged 3 years to 7 years and at 20–30 days post-calving, were enrolled in this study. All the cows were checked by transrectal palpation, ultrasound, and vaginal discharge collection. Endometrial cytology and bacteriological samples were then collected using a cytobrush technique. Nine of 82 beef cows (10.97%) had abnormal vaginal discharges and clinical endometritis (CE), and nine of 73 clinically healthy cows (12.32%) had subclinical endometritis (SCE). The agreement among endometrial examination, polymorphonuclear neutrophil percentage (PMN %), and vaginal discharge score (0–3) was moderate (k = 0.48, p < 0.01), whereas that between vaginal discharge score (0–3) and bacteriological finding was poor (k = 0.032, p = 0.51). The agreement between bacterial finding and PMN % was also poor (k = 0.15, p = 0.02). Escherichia coli was the most common bacteria isolated from healthy (14.6 %), CE (38.8 %), and SCE (42.8 %) cows. Moreover, E. coli was the major bacteriological risk factor for SCE occurrence. E. coli and Staphylococcus aureus were the most common risk factors for the occurrence of CE. The reproductive performance of beef cows was insignificantly affected by CE and SCE

α-Linolenic acid supplementation in BioXcell® extender can improve the quality of post-cooling and frozen-thawed bovine sperm

The present study was conducted to determine the effects of supplementing α-linolenic acid (ALA) into BioXcell® extender on post-cooling, post-thawed bovine spermatozoa and post thawed fatty acid composition. Twenty-four semen samples were collected from three bulls using an electro-ejaculator. Fresh semen samples were evaluated for general motility using computer assisted semen analyzer (CASA) whereas morphology and viability with eosin–nigrosin stain. Semen samples extended into BioXcell® were divided into five groups to which 0, 3, 5, 10 and 15 ng/ml of ALA were added, respectively. The treated samples were incubated at 37 °C for 15 min for ALA uptake by sperm cells before being cooled for 2 h at 5 °C. After evaluation, the cooled samples were packed into 0.25 ml straws and frozen in liquid nitrogen for 24 h before thawing and evaluation for semen quality. Evaluation of cooled and frozen-thawed semen showed that the percentages of all the sperm parameters improved with 5 ng/ml ALA supplement. ALA was higher in all treated groups than control groups than control group. In conclusion, 5 ng/ml ALA supplemented into BioXcell® extender improved the cooled and frozen-thawed quality of bull spermatozoa

Testicular evisceration sequel to trauma and its surgical management in a rabbit

The characteristic thin skin of the scrotal sac in a rabbit was torn resulting in traumatic exposure of the right testicle. Bilateral orchiectomy through an open scrotal approach was performed under general anaesthesia. The rabbit was premedicated with Acepromazine (0.5 mg/kg, 0.15 ml) and Flunixin meglumine (1 mg/kg, 0.03 ml,) intra-muscularly. Isoflurane was used for induction at 5% with O2 flow rate at 0.7 L/min and maintenance Isoflurane, 1.5% - 3%, O2 flow rate = 0.7 L/min) of general anaesthesia. Both right and left testicles were removed and the hemiscrotal incision was closed with 4-0 Vicryl, horizontal mattress suture pattern. Post-operative treatments with antibiotic and anti-inflammatory agents were instituted and the client was advised about how to safely manage aggressive behaviour of rabbits towards each other. The surgical site healed without complication and the neutered rabbit recovered fully within 14 days

Hypo-osmotic swelling test modification to enhance cell membrane integrity evaluation in cryopreserved bull semen

The objective of this study was to enhance the hypo-osmotic swelling test evaluation when it reads under light microscope using 5% formaldehyde-fixed sperm solution buffer (FSSB). Twenty four ejaculates were collected from six crossbred bulls using electro-ejaculator (EE). Tris-egg yolk extender was used to cryopreserve the semen. Concentration, volume, motility, morphology and viability rates of fresh semen were evaluated and samples were cryopreserved in liquid nitrogen. After two weeks of liquid nitrogen treatment (freezing), the motility, morphology and viability rate of the semen were evaluated. In order to carry out a hypo-osmotic swelling test, post-thaw semen was divided into four aliquots based on period of incubation (30 or 60 minutes) adding FSSB to half the samples. The components of FSSB were 5% formaldehyde and 1% eosin-nigrosin stain in PBS. Results showed that F (61.48 ±0.89%) resulted in higher percentage (P0.05) with N60 (60.90±0.70%). In conclusion, adding 10 µl of FSSB after 60 minute of incubation with hypo-osmotic swelling solution (HOSS) will enhance evaluation of the hypo-osmotic swelling test (HOST) under light microscope

Modification of electro-ejaculation technique to minimise discomfort during semen collection in bulls

The aim of this study was to reduce acute discomfortness experienced in bulls during semen collection by electro ejaculation method. The normal electro ejaculation method of semen collection (Method I) was compared to a modified method involving three stages of graduated electrical stimulation (Method II) in four crossbred bulls. The results showed that intensive muscle spasm, bull struggling and arc back were reduced (p < 0.05), as well as the time of penile protrusion (p = 0.003) and semen emission (p = 0.084) were improved using Method II than Method I. However, the total time taken for semen collection was the same in both methods. Also, there were no significant differences in semen parameters such as sperm volume, motility, morphology, viability, and concentration. In conclusion, gradation of electrical stimulation into three stages (our modified Method II) could help to ease the collection of semen samples from bulls with minimum discomfort signs. Furthermore, the modified method is also recommended to use for other animals, in particular, the wild animals

Assessment of three different endometrial cytological sampling methods in postpartum beef cows

The aim of this study was to assess three different cytological endometrial sampling methods used to estimate polymorphonuclear leukocytes (PMN) under high power field (HPF) microscopy and to determine subclinical endometritis in postpartum beef cows. Forty beef cows aged 3-7 years were sampled at week three and four after calving by endometrial cytology methods. The cytological sampling methods used included; cotton swab (CS), cytobrush (CB) technique, and low volume flush (LVF), respectively. The mean PMN counts at the third week was higher (p<0.01) (12.2 cells HPF-1 than on the fourth week (4 cells HPF-1). The average PMN counts using CB alone was significantly higher (11.3 cells HPF-1) than CS (7 cells HPF-1) and LVF ( 6 cells HPF-1) methods. Smears from CB had more endometrial cells (58.55 cells HPF-1) at HPF, which was significantly higher (p<0.01) than CS and LVF methods. Both CB and CS methods yielded more intact cells (62.4 % and 61.9 %) (p <0.01) than LVF (52.4 %). The prevalence of subclinical endometritis in the beef cows between 22 and 28 days postpartum using a threshold value of ≥8 % by cytobrush method was 12.5%, which is considered low. In conclusion, CB method was found to be better and effective technique in comparison to other cytological methods used in obtaining endometrial cytology samples

Effects of EBN on embryo implantation, plasma concentrations of reproductive hormones, and uterine expressions of genes of PCNA, steroids, growth factors and their receptors in rats

This study was conducted to determine the effect of edible bird's nest (EBN) supplement on uterine function and embryo-implantation rate. A total of 24 adult female rats, divided equally into four groups, were treated with different doses of EBN for 8 weeks. In the last week of treatment, intact fertile male rats were introduced into each group (three per group) for overnight for mating. On day 7 post-mating (post-implantation), blood samples were collected from the hearts of anaesthetised rats that were later sacrificed. The uteri were removed for assessment of embryo implantation rate, histological and electron microscopic examination, and immunohistochemical analyses. Results showed that as the concentration of EBN supplemented increased, the pregnancy and embryo implantation rates were also increased in the treated groups; significantly at G3 and G4. Although histological evaluation did not show much difference among the groups, scanning electron microscopic examination showed enhanced development of elongated microvilli and pinopods in G4. Results also revealed up-regulated expressions of epidermal growth factor (EGF), EGF receptor (EGFR), vascular endothelial growth factor (VEGF), proliferating cell nulear antigen (PCNA), and progesterone and estrogen receptors (P4R, E2R) in the uteri of treated groups. Moreover, plasma E2, P4, growth hormone (GH) and prolactin (P) levels were higher (p < 0.05) in G3 and G4. The EBN increased the antioxidant (AO) and total AO capacities (TAC) and reduced oxidative stress (OS) levels in pregnant rats. In conclusion, findings of this study revealed that EBN enhances fertility and embryo implantation rate via promoting proliferation and differentiation of uterine structures as evidenced by the upregulation of the expressions of steroid receptors, EGF, EGFR, VEGF, and PCNA in the uterus. Furthermore, observations of improved growth of ultrastructural pinopods that assist in embryo attachment with uterine epithelium, increased concentrations of E2, P4, GH and P levels, as well as increased AO capacities with reduced OS levels in the treated groups might reflect additional possible mechanisms by which EBN enhances embryo implantation rate and pregnancy success