Influence of alpha linolenic acid on the motility, viability, antioxidant activity and fertility of frozen-thawed New Zealand white rabbit buck semen

[EN] Freezing and thawing processes result in production and accumulation of high concentrations of reactive oxygen species that are detrimental to spermatozoal motility and fertility. Therefore, supplementation of exogenous source of antioxidants to freezing diluent is crucial. The aim of the present study was to investigate for the first time whether supplementation of semen diluent with alpha linolenic acid (ALA) can improve motility, viability, membrane integrity, antioxidant status and fertility of post-thaw rabbit spermatozoa. Semen was collected and pooled from fifteen New Zealand white rabbit bucks. Semen samples were diluted with a tris-citrate-glucose (TCG) extender supplemented with ALA (0, 50, 75 and 100 μmol). Then, extended rabbit semen was cooled at 5°C and cryopreserved in liquid nitrogen. After thawing, spermatozoal quality parameters (individual motility %, viability %, osmotic resistance %, and acrosome integrity %), antioxidant activity (SOD, CAT, and GSH activities), lipid peroxidation (malondialdehyde) and fertility (conception and kindling rates) were evaluated. Results revealed that supplementation of rabbit semen extender with 50 μmol ALA significantly (P<0.05) increased spermatozoal characteristics including motility (56.54%), viability (60.01%), acrosome status (72.66%) and membrane integrity (59.13%). The activity of semen antioxidant enzymes (SOD, CAT, and GSH) showed a significant improvement with a marked decrease in lipid peroxidation. Moreover, the conception (73.30%) and kindling (70.00%) rates were significantly (P<0.05) higher in does inseminated with thawed semen treated with 50 μmol ALA in comparison with other concentrations (0, 75 and 100 μmol). In summary, supplementation of rabbit semen extender with 50 μmol ALA improved motility, viability, membrane integrity, acrosome integrity, antioxidant enzymes activity and fertility of post-thaw rabbit spermatozoa. Our findings suggested that higher concentrations of ALA are detrimental to post-thaw characteristics of New Zealand white rabbit buck spermatozoa. To achieve better results, the semen freezing extender should be supplemented with ALA at lower concentrations, especially 50 μmol.El-Shahat, KH.; Fadl, AM.; Abdelnaby, EA. (2022). Influence of alpha linolenic acid on the motility, viability, antioxidant activity and fertility of frozen-thawed New Zealand white rabbit buck semen. World Rabbit Science. 30(3):219-226. https://doi.org/10.4995/wrs.2022.1704221922630

Correlation between Uterine Hemodynamics, Sex Steroid Hormone Concentrations, and Enzymatic Antioxidant Levels in Postpartum Buffaloes

The present investigation aimed to evaluate uterine hemodynamics in six multiparous postpartum buffaloes and their relationship with sex steroid hormone concentrations and enzymatic antioxidant levels. The buffaloes were examined by transrectal Doppler ultrasonography to record the vascular perfusion in uterine arteries of both ipsilateral and contralateral ones. All Doppler indices such as peak (PV) endpoints (EV) of velocity, peak systolic velocity (PSV), blood flow volume (BFV), resistance (RI), and pulsatility index (PI) were recorded from 1st to 6th postpartum. The blood samples were collected starting from 1st-week post-calving and every week thereafter following each ultrasound Doppler examination for assay of steroid hormones (progesterone and estradiol) and antioxidant (superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase, CAT) were measured. PV of the ipsilateral previously gravid arteries showed a linear pattern of significant (P=0.001) decline from the 1st week after parturition till the 6th week. This decline was also associated with a linear decrease in EV from the 1st week till the 6th week after birth. While contralateral PV and EV are not significantly changed throughout weeks after parturition. The levels of SOD and CAT are significantly elevated at 1st week postpartum compared to the 6th week after parturition. In contrast, the GPx levels did not reveal any significant differences during the puerperal period. Estradiol and progesterone declined from 1st to 5th week after parturition. PV of the ipsilateral uterine artery had a significant (P≤0.05) positive correlation with BFV (r=0.49), estradiol 17- α(r=0.98) and progesterone (r=0.85). The same parameter showed a statically (P≤ 0.001) positive correlation with SOD (r=0.87) and CAT (r=0.92). While, Ipsilateral uterine RI showed a significant (P≤0.05) negative correlation with PV(r=-0.85), BFR ((r=-0.62), estradiol 17- α(r=-0.52), and progesterone (r=-0.88), in addition, RI also correlated negatively with both SOD (r=-0.57) and CAT (r=-0.63). Progesterone and estrogen levels are strongly correlated with SOD and CAT. The uterine hemodynamics in buffaloes is affected by the day of the postpartum period. SOD and CAT antioxidants recorded herein, except GPx, increase in the 1st weeks of calving and are affected by the day of the postpartum period

Evaluations of Ovarian and Luteal Blood Flow Waveform Patterns in Buffalos Subjected to OvSynch Protocol in Cold and Hot Seasons

This current study aimed to determine ovarian and luteal blood flow waveform patterns in buffalos synchronized using OvSynch protocol in cold and hot seasons. Six cyclic buffalo cows aged 6±0.5 years old, having a weight of 400 ± 50 kg, were scanned daily along three successive estrous cycles transrectally by Doppler ultrasonography to evaluate the normal ovarian hemodynamic during the normal spontaneous ovulation and before the start of experiments. Buffaloes were synchronized with gonadotropin[GnRH] –prostaglandin[P] –gonadotropin (GPG) protocol in which animals received 10μg of GnRH on day ??, 0.250μg of PGF2α on day 7, and another dose of 10μg of GnRH was administered 48h after the PGF2α injection. Blood sampling and ovarian ultrasound examinations (color and spectral Doppler modes) were conducted on the day of the estrous and luteal phases. Results revealed that peak systolic velocity waveform (PSV) was significantly (P&lt;0.05) increased in the cold season compared to the hot season. The Luteal blood flow after the end of OvSynch protocol on days (5,7,9, and 11) was significantly increased in the cold season than that in the hot one. The serum levels of estradiol (E2) and nitric oxide (NO) after the second GnRH injection in the OvSynch protocol were significantly (P&lt;0.05) elevated in the cold season as compared to the hot one. Moreover, the progesterone (P4) levels had risen in OvSynch-treated buffaloes on days 5,7,9, and 11 of the cycle in the cold season compared to the hot one. Conclusion: In the cold season, ovarian hemodynamics was significantly improved compared to the hot one; this may influence the reproductive efficiency of buffaloes. Further studies were needed to prove it

Comparison Between Mediastinum Thickness, Hormonal Levels, Nitric Oxide, and Testicular Hemodynamics in Baladi Bucks at Prepubertal and Postpubertal stages

This study aimed to compare the testicular morphometry, mediastinum thickness, hormonal levels, hemodynamic, echogenicity, and heterogeneity in Baladi bucks at prepubertal and postpubertal stages. Five bucks (Capra hircus) were evaluated in two different stages of growth: prepubertal (age 4.5± 0.6 months; 15.0 ± 3.0 kg) and post-pubertal stages (age 13.0 ± 1.3 months; 33.0 ± 2.5 kg). Scrotal circumference, testicular dimensions, mediastinum thickness, echogenicity, heterogeneity, Doppler parameters, semen collection, testosterone, estradiol, follicle-stimulating hormone (FSH), luteinizing hormones (LH), and nitric oxide metabolites (NOMs) were measured. There was an (P&lt;0.05) elevation of the testicular length, width, and scrotal circumference. Mediastinum thickness and colored areas toward and away from probe were increased (P&lt;0.05) in post-pubertal age (2.18±0.01, 6556±32.58, and 7845±65.44) compared to pre-pubertal one (1.27±0.96, 4290±42.12, and 5144±54.24). The spectral graph was characterized by low resistance (RI), moderate pulsatility (PI) with high peak velocity, and low endpoints in the post-pubertal stage while the endpoint was equal to zero in young bucks. The post-pubertal age was associated with a marked decline (P&lt;0.05) in echogenicity, heterogeneity, and RI, while estradiol, testosterone, and NOMs levels were increased (P&lt;0.05). It could be concluded that the post-pubertal stage in Baladi bucks is associated with changes in testicular width, length, mediastinum thickness, RI, scrotal circumference, echogenicity, pixel heterogeneity, testicular colored area away and toward the probe, end-diastolic point, testosterone, nitric oxide, and estradiol levels, as all those variables are considered an accurate markers for the onset of sexual maturity in Baladi bucks. 

Coat color affects the resilience against heat stress impacts on testicular hemodynamics, reproductive hormones, and semen quality in Baladi goats

Abstract Drastic climatic changes threaten animal productivity and prolificacy, whose adaptability is governed by its pheno- and genotypic traits. This study was aimed at investigating the effect of coat color on the adaptability of goat bucks under heat stress conditions from the perspectives of testicular blood flow (TBF) and biometry, reproductive hormones, and semen quality. Twenty bucks (Capra hircus) bearing different coat colors were selected from a large flock and divided into four equal groups (n = 5 each) as follows: black coat (BC; 100% black), brown coat (BrC; 100% dark brown), white coat (WC; 100% white), white-black coat (WBC; 50–60% white). Bucks were examined for TBF [Doppler ultrasonography and serum nitric oxide (NO)], testosterone (T) and luteinizing hormone (LH), seminal plasma oxidative biomarkers [catalase (CAT), total antioxidant capacity (TAC), and malondialdehyde (MDA)], and sperm traits percentages [progressive motility (PM), viability (SV), normal morphology (NM), and sperm concentration (SC) once a week for seven consecutive weeks (W1-W7) in the summer season (temperature humidity index = 88.4–92.2). Specifically, at W3-W7, darker bucks (BC and BrC) testicular volume, testicular colored area, T, NO, CAT, TAC, PM, SV, NM, and SC (W7 only) differed significantly (P < 0.05) by decrease than the lighter ones (WC and WBC). Both Doppler indices and serum MDA concentrations were elevated (P < 0.05) at W3-W7 in the BC and BrC bucks compared to WC and WBC groups. In conclusion, bucks with lighter coats were more resistant to the negative effects of HS on TBF, seminal oxidative biomarkers, and semen quality

Relationship between Main Testicular Hemodynamics and Computer-assisted Analysis in Prepubertal Age for Breeding Selection in Baladi Bucks

This study aimed to determine the relationship between testicular hemodynamics and assisted analysis in pre-pubertal bucks at age of 4-6.5 months. Baladi bucks (n=5) weighting (13-15 kg) and aging (4 months) were examined once a week from week 16 till week 26 of age. Buck’s body weight, scrotal circumference, testicular volume, mediastinum thickness, Doppler, and image analyses were evaluated. Both scrotal circumference and testicular volume were increased (P&lt;0.05) especially in buck 4 followed by buck 2. Both testicular echogenicity (TE) and pixel heterogeneity (PH) showed an increase (P&lt;0.05) in buck 4 then buck 2. Plexus colored area showed a pattern of an abruptly increase either toward or away from the probe (P&lt;0.05) from week 23 of age without marked differences were shown between all buck testes. Both pulsatility (PI) and resistance indices (RI) showed a marked significant (P&lt;0.05) decrease in buck 4 followed by buck 2 from week 23. In addition, testicular peak systolic velocity (PSV) showed a marked linear increase at week 23. There was a positive correlation between TE and PH and both indices. In conclusion, the assessment of testicular hemodynamics and assisted analysis has added a power to the pre-pubertal bucks’ reproductive performance as both tools could help in breeding selection

Incorporation of L-Carnitine in the OvSynch protocol enhances the morphometrical and hemodynamic parameters of the ovarian structures and uterus in ewes under summer climatic conditions

Abstract Heat stress negatively impacts the reproductive performance of sheep including the efficiency of estrous synchronization regimens. This study aimed to investigate the potential effects of L-Carnitine (LC) administration on the efficacy of the OvSynch protocol in ewes under summer climatic conditions. Ewes were synchronized for estrus using the OvSynch protocol and a dose of LC (20 mg/kg body weight) was intravenously (IV) administered on the same day of PGF2α injection to one group (n = 8; LC group), while other ewes (n = 8; control group) received the same protocol without LC. Ultrasonographic evaluation (including B-mode, color, and pulsed Doppler) was used to assess the morphometrical and hemodynamic parameters of ovarian structures [number, size, and blood flow of follicles (GFs) and corpora lutea (CLs)] and uterus during the estrous phase (Day 0), and on Day 8 post ovulation (luteal phase). Uterine artery blood flow (MUA) was assessed by measuring the resistive index (RI) and pulsatility index (PI) at both stages. The serum samples were collected to measure the concentrations of estradiol (E2), progesterone (P4), and total antioxidant capacity (TAC) using commercial kits. Results revealed a significant (P<0.05) increase in the colored pixel area of GFs and uterus in the LC group (392.84 ± 31.86 and 712.50 ± 46.88, respectively) compared to the control one (226.25 ± 17.74 and 322 ± 18.78, respectively) during Day 0. Circulating E2 and TAC levels were significantly (P<0.05) higher in the LC-treated ewes (31.45 ± 1.53 pg/ml and 1.80 ± 0.13 mM/L, respectively) compared to those in the control ewes (21.20 ± 1.30 pg/ml and 0.98 ± 0.09 mM/L, respectively) during Day 0. Moreover, LC improved the colored pixel area of CLs (2038.14 ± 102.94 versus 1098 ± 82.39) and uterus (256.38 ± 39.28 versus 121.75 ± 11.36) and circulating P4 (2.99 ± 0.26 ng/ml versus1.67 ± 0.15 ng/ml) on Day 8. Values of RI of MUA were significantly lower in the LC group compared to the control one on Day 0 and Day 8 (0.48 ± 0.03 versus 0.72 ± 0.03 and 0.58 ± 0.03 versus 0.78 ± 0.02, respectively). In conclusion, LC incorporation in the OvSynch protocol enhanced the morphometrical and hemodynamic parameters of the ovarian structures and the uterus concomitantly with improvements in the TAC, E2, and P4 concentrations in ewes under hot summer conditions

A Ovsynch Produced Larger Follicles and Corpora Lutea of Lower Blood Flow associated Lower Ovarian and Uterine Blood Flows, Estradiol and Nitric Oxide in Cows : Ovsynch ovarian response and blood flow of cows

The use of ovsynch and its modifications for synchronizing estrous cycle and ovulation in ruminants is widely used with lowered conceptions rates. This study aimed to investigate the follicular (F1) and the corpus luteum (CL) hemodynamics, ovarian (OvA) and uterine arteries (UtA) blood flow volumes (BFV) and dynamics associated with the circulating estradiol (E2), progesterone (P4), glucose and nitric oxide (NO) in cows treated with the ovsynch protocol. Eight Friesian cows underwent trans-rectal Doppler scanning and blood sampling each other day throughout two successive non-treated (Spontaneous) and two treated successive estrous cycles (ovsynch). The results revealed that the existed dominant follicles (F1) area, antrum area, color area, and the CL area and the color area on the ovaries declined following the first dose of the gonadotropin-releasing hormone (GnRH, Day-11) till Day -5 then another dominant follicle started growth and reached ovulation (Day 0). Ovsynch pre-ovulatory phase (Day-5 to 0) had more (P&lt;0.01) small, medium, and total follicles. The ovsynch F1 (P=0.001) and the CL (P=0.006) had higher areas but lower color areas percent. The ovsynch increased ipsi-lateral OvA pulsatility index (PI) that associated decreased diameter and time average mean velocity (TAMV) and blood flow volume except Day 6 (BFV, P&lt;0.05). The ovsynch improved (P&lt;0.01) the ipsilateral UtA PI but lowered its diameter, peak systolic velocity (PSV), TAMV, and BFV except Day 10. Ovsynch ovulation was characterized by low E2, NO, and high glucose but the late luteal phase had high (P&lt;0.001) P4 and glucose with low E2. In conclusion, the decreased follicle and luteal vascularization with lowered uterine blood flow and estradiol may adversely affect the quality of the oocyte and the decreased progesterone from Day 7 till Day 10 and the ipsilateral uterine artery BFV may not support the implantation and disturb the maintenance of the embryo after timed insemination

Relationship Between Testicular Morphometry, Vascular Waveform Pattern, Semen Picture, and Specific Serum Testosterone Levels in Baladi bucks Approaching Puberty

 This study aimed to determine testicular blood flow with semen imaging and describe accompanying variations in mediastinal thickness (MT), serum testosterone, and semen characteristics in Baladi bucks approaching puberty and sexual maturity. From 5–10 months of age, Baladi bucks (n = 5) underwent B-mode imaging, Doppler scanning, and blood sampling for testosterone assaying once a month. From 7 months, semen was collected and evaluated. The first semen was collected at a mean age of 7.2 months, while the first spermatozoa appeared at 8.3 months, and the mean age of sexual maturity was 9.4 months. The highest semen volume (1.02 ± 0.09 mL), motility (mass score = 5 and individual = 78.00 ± 1.58%), morphology (94.74 ± 1.99%), alive% (85.21 ± 1.32), and semen concentrations (5.24 ± 0.32 109/mL) were noted in buck 4 (9.5 months; 25.0 kg) followed by others. Testicular width (TW) and MT were positively correlated (r = 0.71; P = 0.03) and increased from 8–10 months (P &lt; 0.05). The spectral graph of young bucks revealed an absent end velocity point. Testicular flow expressed by colored area/pixels was positively correlated with age (r = 0.855), accompanied by increased testosterone levels. Semen characteristics and Doppler parameters were not significantly correlated, except Doppler indices negatively correlated with progressive motility percentage. In conclusion, only the testicular Doppler indices are useful for evaluating testicular function and selecting bucks for breeding since these were the only variables negatively correlated with progressive motility percentage. The spectral Doppler velocities are not useful in estimating the full picture of semen quality in sexually mature bucks. These values should be assessed cautiously since many alterations could lead to the elevation or decline of testicular Doppler parameters.

Eco-friendly Synthesized Zinc Oxide Nanoparticles Improved Frozen-thawed Semen Quality and Antioxidant Capacity of Rams

Zinc oxide nanoparticles (ZnO-NPs) have antimicrobial, antioxidants, and anticancer properties. This study aimed to improve the post-thawing semen characteristics of rams using three concentrations of ZnO-NPs. ZnO-NPs in 0.0, 0.5, 1.0, 1.5 mg/ml tris-based diluents were used to extend semen collected from five Awassi rams twice weekly for two months. Post-thaw semen evaluation and measurement of Malondialdehyde (MDA), nitric oxide (NO), ascorbic acid (AA), superoxide dismutase (SOD), glutathione peroxidase (GPx), total cholesterol (CHO), low density lipoproteins (LDL), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), zinc, and copper (Cu). With increasing ZnO-NPs concentrations, the post-thaw sperm total motility (STM %), progressive motility (SPM) %, viability (SV%), functional membrane integrity (FMI %), acrosome integrity (AI %), NO, LDH, ALP, Cu, and zinc ascended linearly (P≤0.0001) but the abnormal sperm (AS%) and SOD decreased (P≤0.0001). The addition of 1.0 mg/ml ZnO-NPs increased (P≤0.0001) GPx but decreased (P≤0.0001) CHO and LDL. All concentrations of ZnO-NPs preserved (P≤0.0001) high AA compared to 0.0mg/ml and did not influence MDA. The addition of 0.5 mg/ml ZnO-NPs reduced (P≤0.059) Cu concentrations. All semen characteristics had strong positive correlations with zinc, LDH, and ALP and negative correlations with SOD. AS % had negative correlations with zinc, ALP, and LDH and positive correlation with SOD. All the three added concentrations of ZnO-NPs improved the post-thaw semen motility, viability, fertilizing capacity, and antioxidant capacity but 1.5mg/ml semen diluents proved the best concentration for semen preservation