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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 On day 9, IVD was withdrawn, and 0.5mg PGF2α (Estrumate®, Intervet, MSD Animal Health) plus eCG (500 IU, Folligon®, Intervet, MSD Animal Health) was given im.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 (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% (p0.05). 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

Progressive individual motility is the main parameter used to determine the reproductive potential of a buffalo bull. This information contributes to deciding to use and process or not the ejaculate obtained and therefore assume that the spermatozoa from the seminal sample could achieve pregnancy in inseminated buffaloes. The association of sperm motility to fertility and/or pregnancy rate (PR) has yielded controversial results. To contribute to knowledge in buffalo reproduction, an experiment was carried out where four sperm motility descriptors (VCL: curvilinear velocity, VSL: straight-line velocity, VAP: average path velocity, and LIN: linearity) were evaluated using a Computerized Semen Analysis System (Sperm Class Analyzer®, CASA System, Barcelona, Spain) in samples obtained from 8 frozen-thawed straws of 8 Mediterranean buffaloes. The PR data for each buffalo bull were obtained from 2 farms where the management and FTAI protocol employed was handled by the same professionals and straws of similar provenance and proper nitrogen thermos management were used. The PR data for each buffalo bull was confirmed by ultrasonography, organized in Excel® and analyzed with SAS®, associating VAP to PR, using a Chi-square analysis. To achieve the objective, the PR obtained was categorized from highest to the lowest value (A: PR>70%, B: PR>60≤70%, C: PR>50≤60%, D: PR>40≤50%), in addition to creating sperm subpopulations (Sp) based on the quality of sperm movement, for which it was decided to use the VAP, since it is a variable that integrates the VCL and the VSL and indirectly assumes the LIN, creating the following categories: Z= Sp with VAP value ≥ 80 mμ/s, Y: Sp with a VAP value ≥ 50 and < 80 mμ/s, X= Sp with a VAP ≥30 and < 50 mμ/s, W=Sp with a VAP ≥ 10 0.01). There is an evident association between the average speed (VAP) provided by the CASA system and the PR in this species. This type of analysis provides the opportunity to quantify sperm movements and group them to generate more accurate sperm motility values, which allows the prediction of the fertility and/or PR in this species.

A retrospective study (2016-2018) was conducted at the commercial farm “La Fortuna”, located south of Lake Ma-racaibo, Zulia state, Venezuela, to analyze the effect of fixed-time artificial insemination (TAI) and natural mating (NM) on birth weight (BW), weaning weight (WW), age at weaning and post-weaning weights in dairy buffalo calves. The effect of buffalo calf sex on these variables was also assessed. After birth, calves were kept with their mother for 15 days. After that, they were suckled twice daily until weaning and went daily to the pasture from 30 days postpartum. During the study period, 15 AI and ten natural mating bulls were used. Data were analyzed by the general linear model of SAS. BW was greater in calves born from TAI (n=141; 36.2 ± 0,1 kg) than from NM (n=131; 35.7 ± 0.2) (p=0.0320). The age at weaning was similar between breeding methods (TAI: 285.1 ± 3.7 d; NM: 290.9 ± 3.8 d; p=0.2731). Weaning weight was ∼ 10 kg greater in calves born from TAI (178.8 ± 3.8 kg) than from NM (168.6 ± 4.0 kg) (p=0.0450). Adjusted weight at 205 (202.1 ± 4.6 vs. 179.2 ± 4.7 kg respectively; p=0.0006) and 365 days (248.2 ± 6.1 vs 230.8 ± 6.3 respectively; p=0.0466) was significantly greater in buffaloes resulting from TAI than from NM. Female buffaloes (n=157) were born lighter than males (35.7 ± 0.1 vs 36.2 ± 0.2 kg; p=0.0500). The former (295.8 ± 3.5) reached an older age at weaning than the latter (280.2 ± 4.1 d) (p=0.0041). The weight at weaning was similar between sexes (174.9 ± 3.6 and 172.6 ± 4.2 kg respectively; p=0.6838). Adjusted weight at 205 days was greater in females (198.6 ± 4.3 kg) than in males (182.7 ± 4.3) (p=0.0178); however, the weight at 365 days did not vary between the sexes (females: 243.2 ± 5.7 kg; males: 235.8 ± 6.3 kg; p=0,3663). This study demonstrated that artificial insemination is a valuable tool for improving weaning and yearling weights in buffalo herds. Although female buffalo calves were born at lower birth weights and weaned at older ages, they achieved better post-weaning weights.

Fixed-timed artificial insemination (FTAI) protocols combining estradiol (E2) and progesterone (P4) are commonly used in the tropics, with adequate pregnancy rates throughout the year in buffaloes. However, the application of E2 and its related esters in food-producing animals is banned in the European Union and by the FDA-USA. This study aimed to evaluate the pregnancy rate (PR) after FTAI in buffalo cows (Bubalus bubalis) treated during the breeding (September-January) and non-breeding season (February-June) using a protocol based on P4 intravaginal devices (IVD) combined with GnRH and PGF2α. The trial was performed on a commercial buffalo farm in Zulia State, Venezuela. Two hundred forty-seven buffalo cows (1-14 calvings) with a body condition score (BCS) of 3.15 ± 0.39 were selected. Cows were synchronized on a random day of the estrus cycle. To synchronize the ovulation on day 0, a dose of 10.5 μg GnRH-analog (Buserelin benzoate acetate, Gestar®) was injected intramuscularly (IM) plus the insertion of an IVD maintained for nine days [TRIU B®: 1.0 gr P4, (n=139); CIDR®: 1.38 gr P4 (n=77); or PREGNAHEAT-E®: 250 mg of medroxyprogesterone acetate (MPA), (n= 18)]. On day 9, IVD was removed, and 0.50 mg of Cloprostenol (Estrumate®, Intervet, MSD Animal Health) plus 500 IU of eCG (Folligon®, Intervet, MSD Animal Health) were given IM. On day 11, a dose of 10.5 μg GnRH-analog IM was used, and 8-12 hours later, they were artificially inseminated using frozen-thawed semen of bulls with proven fertility. The pregnancy rate (PR) was determined 30-32 days after AI by ultrasound. The data was analyzed by logistic regression through the Proc Logistic, and continuous and linear character variables were analyzed through the Proc GLM of the SAS® package. The overall PR was 52.2% (n = 129/247). Evaluation of independent variables, such as the number of calving and BCS, did not affect the PR (p>0.05). The PR was similar (p>0.05) amongst the reproductive and non-reproductive seasons [52.2%: (66/129) vs. 53.4% (63/118), respectively]. IVD-type did not affect the PR (p>0.05) [TRIU- B®: 52.6% (80/152), CIDR®: 53.3% (41/77) and PREGNAHEAT-E®: 44.4% (8/18)]. In conclusion, this FTAI protocol based on P4 and GnRH and PGF2α produced adequate pregnancy rates during breeding and non-breeding seasons. These findings suggest that GnRH could be considered to substitute E2 esters in the P4-based FTAI protocols in water buffaloes under tropical conditions.

The livestock industry has used genetic selection to improve phenotypic characteristics for decades. The identification of characteristics in the population, such as adaptation, disease resistance, production capacity, and specific phenotypic characteristics, combined with the use of directed and assisted reproduction techniques, have been the tools that have allowed us to reach current production levels in the livestock industry. Since the discovery of DNA and the advancement of molecular genetics, it is well known that phenotypic characteristics (milk production, meat production, and absence of horn, among many others) are conditioned and governed mainly by DNA. Thus, indirectly, genetic improvement programs based on the measurement of the characters of interest, selection, and control of reproduction to multiply the selected-desired character have as their principle the intervention and control of the DNA replicated in the next generations. On the other hand, genomic sequencing is the technology that has advanced the fastest in the last decade. Its applications and benefits for human development and improving the quality of life of current and future generations are innumerable and have tremendous implications. For instance, genomic selection has been proposed as a valuable tool to combat climate change. In this sense, genomic selection uses information from many genetic markers or single nucleotide polymorphisms (SNPs) in conjunction with measurements (phenotypes) of essential traits in livestock to improve selection accuracy, reduce the generation intervals, and increase genetic gains since the observation can be read and estimated with a DNA sequencing before the population express the character even before the animal is born. Therefore, genomic selection has generated significant buzz as it has the potential to revolutionize livestock production globally, allowing farmers to be more productive using fewer resources (feed, water) to fill the rapidly changing climates and human needs.

Insulin-like growth factors (IGFs) are essential for oocyte maturation. Their bioavailability is regulated by their respective binding proteins (IGFBPs) and proteases. IGFBP-4 blocks the biological effects of IGFs. High IGFBP-4 expression has been associated with follicle atresia. We hypothesized that IGFBP-4 affects oocyte developmental competence during maturation. Therefore, the aim of this study was to examine the effect of IGFBP-4 on the developmental rate of bovine cumulus–oocyte complexes (COCs) during in vitro embryo production. Abattoir-derived COCs were matured with rbIGFBP-4 (2000, 540, and 54 ng/mL) compared to a control. Cumulus expansion, oocyte maturation, cleavage, blastocyst, and hatching rates were evaluated. Furthermore, blastocyst gene expression of SOCS2, STAT3, SLC2A1, SLCA3, BAX, and POU5F1 transcripts were quantified using RT-qPCR. No statistical differences were detected among the groups for cumulus expansion, maturation, cleavage, blastocyst rates, or all gene transcripts analyzed. However, at day 8 and 9, the number of total hatching and successfully hatched blastocysts was lower in 2000 ng/mL rbIGFBP-4 compared to the control (day 8: total hatching: 17.1 ± 0.21 vs. 31.2 ± 0.11%, p = 0.02 and hatched blastocyst 6.7 ± 0.31 vs. 21.5 ± 0.14%, p = 0.004; day 9 total hatching 36.4 ± 0.18 vs. 57.7 ± 0.10%, p = 0.009 and hatched blastocyst 18.2 ± 0.21 vs. 38.1 ± 0.11%, p = 0.004). We concluded that high concentrations of rbIGFBP-4 might negatively affect the subsequent ability of the embryo to hatch and possibly compromise further elongation.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10-11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode's Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10-11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

Para evaluar el efecto del protocolo Ovsynch sobre parámetros de fertilidad en vacas mestizas doble propósito en anestro y definir el momento óptimo para la IA a tiempo fijo (IATF), 37 de 48 vacas acíclicas se asignaron a los siguientes grupos: T1: día 0: GnRH; día 7: PGF2α día 9: GnRH (Ovsynch); + IATF 24 h post-última inyección de GnRH, (n=14); T2: Ovsynch + IATF 16 h post-última inyección de GnRH, (n = 11); C: control, (n = 12). Las variables estudiadas fueron: Tasa de Estros Prematuros (EP), Tasa de Concepción (TC), Tasa de Preñez (TP) e Intervalo Tratamiento-Preñez (ITP). Las variables EP, TC y TP fueron medidas con el procedimiento PROC FREQ, del paquete estadístico SAS; mientras que la variable ITP se analizó mediante el procedimiento lineal general PROC GLM del SAS. La tasa de EP fue mayor (P < 0,05) en Ovsynch (30,5%, 11/36) vs C (8,3%, 1/12). La TC fue similar para EP (72,7%, 8/11) y T1 (42,8%, 6/14); pero T1 presentó una TC mayor (P=0,06) en comparación con T2 (42,8%, 6/14 vs 9,1% 1/11). No fueron encontradas diferencias para la TP en T1 vs T2 (50%, 7/14 vs 45,5%, 5/11), pero ambos grupos presentaron mayor TP comparados con C (25%, 3/12) (P < 0,05). El ITP fue menor (P < 0,05) en T1 (38,9 + 15,7 días) comparado con T2 y C (62,4 + 18,8 días y 60,0 + 35,1 días respectivamente). En conclusión, el tratamiento Ovsynch +IATF 24 h posterior a la última inyección de GnRH resultó en una mayor TC y acortó el ITP en vacas mestizas en anestro. Se demostró que el protocolo Ovsynch representa una alternativa para el control del anestro postparto y mejoramiento de la eficiencia reproductiva de las ganaderías bovinas de doble propósito.

Para evaluar el efecto de los días postparto al tratamiento (DPP), predominio racial (PR), número de partos (NP) y época del año (E) sobre la ciclicidad y fertilidad de vacas mestizas en anestro tratadas con esponjas intravaginales (EI) impregnadas con Medroxi-acetato progesterona (MAP) más una inyección de eCG y PGF2a; se condujo un experimento en una finca ubicada en un bosque húmedo-tropical en el municipio Machiques de Perijá-Zulia, Venezuela. Se seleccionaron 72 vacas mestizas Bos taurus í— Bos indicus primíparas (47) y multíparas (25) entre 45 y 108 días postparto (DPP) y condición corporal (CC) entre 3 y 3,5. Las vacas recibieron el siguiente esquema hormonal: Día 0: inserción de la EI, inyección de 5 mg de 17 B-Estradiol y 50 mg de MAP im; Día 6: 500 UI de eCG y 25 mg de PGF2a im; Día 8: retiro de la EI; Día 9: 1 mg de 17 B-Estradiol im. Un grupo fue tratado antes de los 70 DPP (MAP < 70DPP; n = 25) y otro después de los 70 DPP (MAP > 70DPP; n = 47). Se estudió la tasa de sincronización (TS), fertilidad al primer servicio (F), fertilidad global (FG), Intervalo Tratamiento-Concepción (ITC), Intervalo Parto-Concepción (IPC) y periodo de sincronización (PS). Las variables TS, F, y FG se analizaron con PROC LOGISTIC, SAS; mientras que ITC, IPC y PS con PROC GLM, SAS. No se encontró efectos del PR y NP sobre la TS, F, FG, ITC, IPC y PS. Las vacas con PR Bos indicus presentaron mayor F y FG en el grupo MAP < 70DPP comparada con MAP > 70DPP (F: 81,8 vs 43,7%; FG: 84,6 vs 45,5%). La TS fue mayor en las vacas tratadas en la época lluviosa comparada con la seca (96,4 vs 69,8%). La F solo fue mayor en la época lluviosa cuando las vacas fueron tratadas antes de 70DPP vs MAP > 70DPP (87,5 vs 42,1%). El PS fue menor en la época seca (36,9 h) vs la época lluviosa (48,8 h). El IPC fue menor en MAP < 70DPP (95,8 días) comparado con MAP > 70DPP (148,3 días). En conclusión, los días postparto al momento de la aplicación de la EI fue el factor que más tendió a afectar la respuesta al tratamiento.