Evaluation of dietary arginine supplementation to increase placental nutrient transporters in aged mares.

Nine pregnant mares (18.2 ±â€…0.7 yr; 493.82 ±â€…12.74 kg body weight [BW]) were used to test the hypothesis that dietary supplementation of l-arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and assigned randomly to dietary treatments of either supplemental l-arginine (50 mg/kg BW; n = 7) or l-alanine (100 mg/kg BW; n = 6; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal Bermudagrass hay. Treatments began on day 14 of gestation and were terminated at parturition. Mare BW, body condition score (BCS), and rump fat were determined, and body fat percentage was calculated every 28 d and concentrate adjusted accordingly. Doppler blood flow measurements including resistance index (RI) and pulsatility index for uterine artery ipsilateral to the pregnant uterine horn were obtained beginning on day 21 and continued every 7 d until day 154 of gestation, and prior to parturition. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn was analyzed histologically to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (SLC7A1) proteins. Semiquantitative analyses were performed using 10 nonoverlapping images per sample fixed in a 10× field (Fiji ImageJ v1.2). Mare performance data were analyzed using PROC MIXED in SAS and foaling and placental data were analyzed using PROC GLM. Gestation length at parturition was not influenced (P > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P < 0.01) and greater percent body fat (P = 0.03), and BCS (P < 0.01) at parturition. Arginine-supplemented mares had a lower RI than control mares prior to parturition (P < 0.01). Body length, height, and BW of foals at birth, as well as placental weight and volume, and immunohistochemical staining for VEGF and SLC7A1 at parturition, were not affected (P > 0.05) by maternal arginine supplementation. These results indicate that dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares. A larger number of mares is required to extend knowledge of effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Inclusion of supplemental antibiotics (amikacin - penicillin) in a commercial extender for stallion semen: Effects on sperm quality, bacterial growth, and fertility following cooled storage.

In this study, the effectiveness of supplementing INRA-96® extender (INRA-Control; original antibiotic formulation: potassium penicillin G = 38 µg/mL; gentamicin sulfate = 105 µg/mL; amphotericin B = 0.315 µg/mL) with amikacin sulfate and potassium penicillin G (AP) was determined. In Exp. 1, two sources of amikacin (INRA-AP-Sigma or INRA-AP-GoldBio) in combination with penicillin G were compared with ticarcillin/clavulanate (INRA-Tim) or no-supplemental antibiotics (INRA-Control) to examine effects on sperm quality and commensal bacterial growth. No differences were detected in semen quality among treatments after 30 min of exposure (Time 30min) or 24 h of cooled storage (Time 24 h; P > 0.05). At both time periods, commensal bacterial growth was significantly lower in Groups INRA-AP-GoldBio and INRA-AP-Sigma than in INRA-Tim or INRA-Control (P < 0.05). In Exp. 2, increasing doses of amikacin sulfate (GoldBio) plus potassium penicillin G (Sigma) - AP (AP-1000, 2000, 3000, 4000 or 5000 µg-IU/mL, respectively) were added to INRA-96® extender and their effects on sperm quality and commensal bacterial growth were evaluated at Time 30min and Time 24 h. Slight reductions in progressive motility and viability were observed at Time 30min in Groups AP-4000 and AP-5000 as compared to other treatment groups (P < 0.05); however, no differences in sperm quality were detected among treatment groups at Time 24 h (P > 0.05). At both time periods, commensal bacterial growth was significantly lower in Groups AP-3000, AP-4000 and AP-5000 than in AP-1000 and AP-2000 (P < 0.05). In Exp. 3, a breeding trial was conducted to determine the effect of adding a high dose of AP (AP-5000) to INRA-96® extender on resulting pregnancy rates of mares bred with cool-stored semen (Time 24 h). Numerical, but not statistical differences, were observed in pregnancy rates between the mares bred with INRA-Control (6/11; 55%) or INRA-AP-5000 (9/11; 82%; P > 0.05). Supplementation of INRA-96® extender with two different concentrations of AP (AP-1000 or AP-5000) was tested in two clinical cases of stallions where semen was moderately to heavily contaminated with Pseudomonas aeruginosa, or both Klebsiella pneumoniae and Pseudomonas aeruginosa. In both cases, addition of AP resulted in a considerable decrease on bacterial growth in cool-stored semen when compared to the use of the original INRA-96® extender without supplemental antibiotics. In conclusion, the addition of amikacin sulfate and potassium penicillin G to INRA-96® extender allowed for effective control of commensal bacteria without affecting sperm quality. Higher doses of amikacin and penicillin can be safely added to INRA-96® extender to improve the antibacterial activity of this extender against commensal, and potentially pathogenic bacteria, while sperm quality and fertility of cooled semen remains unaffected. Based on the results of the present study, we currently recommend that INRA-96® extender can be safely supplemented with amikacin/penicillin by using a conventional dose of 1000 µg/mL - 1000 IU/mL as a prophylactic measure in cases where contamination of the ejaculates with commensal bacteria is evident. Alternatively, a high dose (5000 µg/mL - 5000 IU/mL) can be used as a control method for potentially pathogenic bacteria.

Influence of L-arginine supplementation on reproductive blood flow and embryo recovery rates in mares.

Supplementation with L-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to L-arginine supplementation or control groups. Mares were fed L-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.

Oral L-arginine supplementation impacts several reproductive parameters during the postpartum period in mares.

L-arginine is an amino acid which can alter pituitary function and increase blood flow to the reproductive tract. The objective was to determine the effect of supplementing 100g of L-arginine on plasma arginine concentrations, follicular dynamics and ovarian and uterine artery blood flow during the estrus that occurs subsequent to foaling. In Experiment 1, mares were fed 100g L-arginine for 1 day during the last 3 weeks of pregnancy and plasma samples taken for every hour for the first 4h and every other hour until 12h.L-arginine supplementation elevated plasma arginine concentrations from 1 to 8h post feeding; arginine peaked at 6h (arginine: 515±33µmol/L; control: 80±33µmol/L). In Experiment 2, mares received either 100g L-arginine or control diets beginning 21 d before the expected foaling date and continued for 30 d postpartum. The reproductive tract was evaluated by transrectal Doppler ultrasonography from Day 1 postpartum through Day 30. There were no differences in ovarian follicular dynamics, ovarian or uterine resistance indices between groups. Vascular perfusion of the F1 follicular wall was greater in L-arginine supplemented mares (37.3±2.6%) than controls (25.4±2.7%; P<0.05). L-arginine supplemented mares had a smaller uterine body and horns and accumulated less uterine fluid than controls (P<0.05). The combination of reducing uterine fluid accumulation, while not altering follicular development, raises the possible use of L-arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success.