Factors influencing pregnancy rate following estradiol-free fixed TAI protocols in water buffaloes
Authors: Camacho de Gutiérrez Adriana , Camacho Obdulio , Fernández Steward , González Yerixo , Gutiérrez-Añez Juan Carlos 
Published: February 2025

Fixed-Time Artificial Insemination (TAI) protocols efficiency is affected by numerous factors. This study evaluated biological- and technical-inherent factors associated with TAI programs. Two hundred forty-seven buffalo cows (1-14 calvings), with a body condition score (BCS) between 2.5 and 4.5, were used. The TAI protocol consisted of: Day 0, an injection of GnRH analog (10.5μg of buserelin benzoate acetate, Gestar®) intramuscularly (im) and one intravaginal device (IVD) for 9 days [TRIUB®: 1.0gr P4, (n=139); CIDR®: 1.38gr P4 (n=77); or PREGNAHEAT- E®: 250mg of medroxyprogesterone acetate (MPA), (n= 18)]. On day 9, IVD was withdrawn, and 0.5mg PGF2α (Estrumate®, Intervet, MSD Animal Health) plus eCG (500 IU, Folligon®, Intervet, MSD Animal Health) was given im. On day 11, the ovulation was induced using GnRH (10.5μg) im. TAI was performed 8-12 h later using semen from Murrah and Mediterranean bulls. Pregnancy diagnosis was performed 30-32 days after by ultrasound. Biological-inherent factors such breed (Murrah, Mediterranean, undefined), postpartum days (PPD; 30–45, 46– 60, 61–90 or ˃90), calving’s number, BCS, milk yield (< 4, 4-6 and ˃ 6 Kg/day), ovarian status at the beginning of the protocol [follicle size (<8 mm, 8-10 mm, >12mm), corpus luteum and cysts], uterine status were studied. In addition, physiological responses at the TAI timepoint, such as tail bending, cervix lubrication, and dilation, were included. More-over, technical-inherent variables such as AI technician, straw size (0.25 and 0.5), time performing AI technique, kind, IVD expulsion of IVDs, and the entry order to AI were evaluated. Data was analyzed by logistic regression using Proc Logistic from SAS®. The protocol showed a similar pregnancy rate (PR; p>0.05) regardless of the season, calving number, BCS, breed, milk yield, follicle size, and PPD period at the time point of a protocol start. Similarly, tail bending, cervix dilation, and lubrication did not influence fertility (p>0.05). However, cows with ovarian cysts at the start of the protocol showed a lower PR than those with CL 22.2 vs. 62.8% (p<0.05) respectively. Furthermore, animals treated with PGF2α and antibiotics to correct abnormal uterine status showed a higher PR 86% vs 50% than animals with apparently normal uterine status. Tech-nical-inherent factors such as IVD type, expulsion of IVDs before the end of treatment, and straw size did not affect the PR (p>0.05). However, when the AI technique was performed in ≤2 minutes, the PR was higher, in comparison to ≥2 minutes (73.1% vs. 42.9%, respectively) (p<0.01). Additionally, docile animals accessing first to the AI showed higher (p<0.05) PR compared to those that struggled and entered last (68.8% vs 48.3%, respectively). In conclusion, this protocol showed adequate PR independent of the season, cows’ breed, and productive status. Moreover, correcting the abnormal uterine status improved the PR and might increase the number of animals to be included in the TAI program. Additionally, technical factors such as time employed to perform the AI and animal behaviour when applying TAI successfully affected the PR and warrant further investigation.