2020) Effect of Moringa oleifera seed extract on antioxidant activity and sperm characteristics in cryopreserved ram semen, ABSTRACT Semen cryopreservation damages sperm due to oxidative stress. This study evaluated the antioxidant capacity of Moringa oleifera seed extract in cryopreserved ram semen and the impact of the extract on sperm characteristics. Semen from eight hair rams (four rams per sampling) was allocated into four groups, according to their treatment prior to cryopreservation: Control (no extract), 0.5 (M0.5), 5.0 (M5.0), and 10.0 (M10) mg/mL of M. oleifera extract. The antioxidant activity (ferric reducing antioxidant power, FRAP) and the spermatic characteristics (sperm viability; progressive motility; fast motility; slow motility; acrosome damage; membrane damage; and mitochondrial activity) were assessed post-thawing. Variables were evaluated with analysis of variance followed by Tukey test. While no significant differences were detected in acrosomal damage, mitochondrial activity, fast-, or slow motility, the antioxidant activity was higher (P < 0.05) in M0.5 and M5 treatments. Viability and progressive motility increased in the M0.5 group (P < 0.05), whereas sperm membrane damage was lower (P < 0.05) in the same treatment. In conclusion, supplementation of ram semen with M. oleifera seed extract enhances antioxidant activity, sperm membrane integrity, viability, and progressive motility after thawing. This suggests that M. oleifera extract could be used as an antioxidant to improve the outcome of semen cryopreservation. Highlights. It is widely known that semen cryopreservation induces sublethal damage to sperm, deteriorating spermatic characteristics, which is largely attributed to oxidative stress. . There are scarce studies regarding the use of plant extracts as a replacement for conventional antioxidants to conserve sperm viability in cryopreserved ram semen. . Addition of Moringa oleifera seed extract to a concentration of 0.5 and 5.0 mg/mL prior to ram semen freezing increased antioxidant activity after cryopreservation. . M. oleifera seed extract at 0.5 mg/mL decreased post-thawing damage to the sperm membrane, increasing both viability and progressive motility. . M. oleifera seed extract could be potentially used as a replacement for conventional antioxidants added to maintain sperm viability in cryopreserved ram semen. ARTICLE HISTORY

To evaluate the effects of season on semen, scrotal circumference and testosterone concentration in Corriente and European-breed bulls, 758 samples were analyzed in four years. Comparisons were made between bulls of the same breed in different seasons and between bulls of different breeds in the same season. Scrotal circumference of both breeds was higher in the summer and autumn (P < 0.01). Sperm concentration, individual motility and live cells were lower in summer and autumn in European-breed bulls (P < 0.01). All percentages of sperm defects were higher, and testosterone was lower in European-breed bulls during summer and autumn (P < 0.01). Gross motility tended to be lower (P = 0.09) in European-based bulls during winter. Scrotal circumference was higher in Europeanbreed bulls than Corriente bulls in all seasons (P < 0.01). Ejaculate volume and sperm concentration were similar in Corriente and European-breed bulls in all seasons (P > 0.1). Individual motility was lower in Europeanbreed than in Corriente bulls in summer and autumn (P < 0.01 and <0.01). Live cells count was lower in European-breed than in Corriente bulls during the summer and autumn (P < 0.01 and <0.01). Major sperm defects were higher for Corriente than European-breed bulls during winter (P < 0.01). Minor cell defects were higher in summer and autumn (P < 0.01) in Europeanbreed than in Corriente bulls. Total defects were lower in Corriente than in European-breed bulls during summer and autumn (P < 0.01), and higher during winter (P < 0.01). Testosterone was higher in Corriente bulls during summer (P < 0.01) and autumn (P < 0.01) than in European-breed bulls. Gross motility was lower (P < 0.05) in European-breed than in Corriente bulls in winter. Season had no effect on most semen characteristics in Corriente bulls, while European-breed bulls had lower semen quality during the hotter seasons. This indicates a better adaptation of Corriente cattle, suggesting that these bulls can be used for mating all year in hot environments.

Background Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples. Methods We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy. Results In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). Conclusions Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.

Trypan Blue (TB) and Brilliant Cresyl Blue (BCB) are used to select and determine viability and competence of oocytes, however, the effects of corpora lutea on cumulus expansion during in vitro maturation are still undetermined. Cumulus-oocytes complexes (COCs) from ovaries with ipsilateral (ICL), contralateral (CCL) and without corpora lutea in either ovary (OCL) were selected by visual examination, TB and BCB staining, and matured in vitro to evaluate cumulus expansion. The overall percentage of visually selected COCs was similar in ICL, CCL and OCL (P > 0.05). Proportions of live and mature COCs were similar in all groups (P > 0.05). The overall percentage of BCB+ COCs was 70.1%. BCB+ ICL COCs had less cumulus expansion (60.5%) than BCB+ CCL and OCL COCs (75.7 and 71.4%, respectively; P < 0.01). BCB− ICL COCs had less cumulus expansion (20.0%) than BCB− CCL and OCL (39.7 and 46.1%; P < 0.01). BCB+ CCL and OCL COCs showed the highest cumulus expansion index (P < 0.01). Presence of corpus luteum in the ovary affects negatively cumulus cells expansion. TB and BCB staining facilitate the selection of oocytes with higher degrees of cumulus expansion.

In beef and dairy cattle, the number of follicular waves affects endocrine, ovarian, and behavioral events during a normal estrous cycle. However, in Mexican-native Criollo cattle, a shortly and recently domesticated breed, the association between wave patterns and follicular development has not been studied. The objective of this study was to evaluate the effect of number of follicular waves in an estrous cycle on development of anovulatory and ovulatory follicles, corpus luteum (CL) development and functionality, as well as estrual behavior in Criollo cows. Ovarian follicular activities of 22 cycling multiparous Criollo cows were recorded daily by transrectal ultrasound examinations during a complete estrous cycle. Additionally, blood samples were collected daily to determine serum progesterone concentrations. Only two- (n = 17, 77.3%) and three-wave follicular (n = 5, 22.7%) patterns were observed. Duration of estrus, length of estrous cycle, and length of follicular and luteal phases were similar (P> 0.05) between cycles of two and three waves. Two-wave cows ovulated earlier (P < 0.05) after detection of estrus than three-wave cows. Detected day and maximum diameter of first anovulatory follicle were not affected (P > 0.05) by number of waves. Growth rate of first dominant follicle was higher (P < 0.05) in three-wave cycles. Onset of regression of the first dominant follicle was earlier (P < 0.01) in cycles with three waves than in those with two waves. In two-wave cycles, ovulatory follicles were detected earlier (P < 0.01) and had lower (P < 0.01) growth rate than in three-wave cycles. Development (i.e., maximum diameter and volume) and functionality (minimum and maximum progesterone concentration) of CL were similar (P > 0.05) between two- and three-wave patterns. In conclusion, Criollo cows have two or three follicular waves per estrous cycle, which alters partially ovulatory follicle development and ovulation time after detection of estrus. Length of estrous cycle, as well as CL development and functionality, was not affected by number of follicular waves.

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Background: Nowadays, Ehrlichia canis receives more attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serologic tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence negative by PCR in blood. Methods: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymphatic nodules, and bone marrow in 59 recently euthanized dogs that had ticks but were clinically healthy. Results: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yield positive results in tissue biopsies as follows: 63.15% from bone marrow, 52.63% from liver, 47.36% from spleen and 15.78% from lymphatic nodules. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). Conclusions: Our results show prevalence of E. canis in tissue from dogs that were negative by PCR in blood. E. canis DNA in tissue was 30% lower in dogs that tested negative in blood samples by PCR, compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in others tissues.

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