The incorporation of Genetics in bovine breeding herds through the use of Artificial Insemination at Fixed Time (TAI) has achieved an increase in profitability by allowing to obtain greater The objective of this review was to compare the performance of programs with natural service, treatment Synchronization before natural service, artificial insemination after oestrus detection and fixed-time artificial insemination (TAI) in beef cattle.
Seminal plasma proteins (SPP) are fundamental for oocyte fertilization by sperm cells. In bovine, the structure and function of SPP have been widely described in several studies, where the spermadhesin family has been highlighted. Spermadhesin proteins are closely related to sperm motility and viability along with protecting the sperm cells against oxidative stress. Spermadhesin-1, also known as acidic Seminal Fluid Proteins (aSFP), exhibits a redox activity that protects the sperm cells from reactive oxygen species (ROS), an important feature that could be taken in advantage to improve the postthaw seminal quality of sperm cells after cryopreservation processes. Therefore, the aim of this research was to produce heterologous aSFP using Escherichia coli as a cell factory. For this purpose, the DNA sequence of aSFP was inserted into a pDAss plasmid, containing a six-histidine tag (6xhis). The obtained construct was used to transform BL21 (DE3) E. coli competent cells. The expression trials were developed at three different temperatures (37°,25°, and 16°C) and two different concentrations of Isopropyl-β-D-1-thiogalactopiranoside (IPTG) as the expression inductor (0.5 y 1.0 mM). The aSFP-H6 produced was purified by affinity chromatography, and the peptide sequence was verified through mass spectrometry. Results evidenced the best conditions for the recombinant aSFP-H6 production (16°C and 1.0 mM of IPTG). In this sense, the viability and the conditions to produce heterologous bovine aSFP were described and could be further used to enhance cryopreservation bovine sperm mediums.
The aim of this work was to describe macroscopically and microscopically the ovaries of alpacas during the fetal stage. We worked with 18 female fetuses collected at the Huancavelica Municipal Slaughterhouse, from alpacas destined for human consumption. The process and analysis of the collected samples was performed at the Faculty of Veterinary Medicine - UNMSM, in Lima, Peru. Fetal age was calculated by measuring the biparietal diameter and divided into three groups: Group I (60 - 150 days), Group II (151 - 239 days) and Group III (240 - 335 days). The weight of the ovaries was 0.02 ± 0.01, 0.03 ± 0.01 and 0.03 ± 0.01 grams in the first, second and third group, respectively. The ovaries were paired, oval-shaped, with a smooth, cream surface, located in the sublumbar region at the level of the 6th and 7th lumbar vertebrae. The cortex and medulla were visible from the third month. Microscopically, in Group I, we observed oogonium, the distinction between cortex and medulla, unilaminar primary follicles, atresia and segmentation of the germ cells. In Group II, we observed preantral primary follicles. In Group III, we observed preovulatory follicles. We concluded that, from day 68 of the fetal stage of the alpaca, ovaries have macroscopic characteristics similar to those of an adult alpaca. Microscopically, they presented ovogonia, primordial follicles, unilaminar primary follicles, preantral follicles, preovulatory follicle, as well as the degeneration of the germ cells.
The aim of the present study was to evaluate the effect of equilibration process on canine sperms after vitrification using coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants. Twelve ejaculates were collected separately by digital manipulation from 12 adult dogs. Only the second fraction of the ejaculate was used in this study, which was evaluated about volume, concetration, viability, total and progressive motility, kinetic parameters and morphology. After evaluation, semen was diluted with a coconut water extender (50% coconut water (v/v), 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% (v/v) and 0.25 M sucrose until final concentration of 100x106 spermatozoa/ml. Samples were divided into three aliquots and each of them was processed at different regimens: without equilibration, 5ºC for 30 minutes and 5ºC for 60 minutes and then vitrified by dropping 30 μl of sperm suspension directly into liquid nitrogen. Sperm pellets were devitrified at least one week later as three of them were dropped into 0.3 mL of CaniPlus AI (Minitüb, Germany), which was previously warmed in a water bath at 37ºC for 2 minutes. Sperm concentration and motility parameters were assayed using a computer-aided sperm analysis (CASA) system, viability-by supravital staining technique and morphology parameters were evaluated in Haemacolor® stained semen samples. In conclusion, our results demonstrate that when vitrification and coconut water extender with addition of 1% soy lecithin and 0.25 M sucrose as cryoprotectants were used, presence of equilibration time of 60 minutes returned the best canine sperm quality results.
The aim of this investigation was to evaluate the effect of the addition of GnRH at the beginning and end of a treatment with progesterone-releasing devices (P 4 ) and estradiol (E2) and the expression of estrus on pregnancy rates (PR) of lactating dairy cows inseminated at fixed time (FTAI). The estrus synchronization treatment consisted on the insertion of a P 4 device for 8 days and the administration of estradiol benzoate (EB) on the day of insertion and estradiol cypionate (ECP) at the time of device removal to induce ovulation. Experiment 1 was designed to determine whether the addition of GnRH at the start of the protocol improved the PR and if the division of the time of artificial insemination (AI) improved PR. The addition of GnRH on Day 0 did not improve PR (44% vs 40%) but the AI of cows showing estrus at 48 h and the administration of GnRH at 48 h and AI at 60 h of those not showing estrus resulted in higher PR than those cows that were FTAI at 48 h, regardless of estrus expression (53% vs 31%; P <0.01). Experiment 2 was designed to study follicular dynamics and assess whether the PR varies using different times of AI (48 h vs 60 h), according to the expression of estrus. The division of AI and adding GnRH at 48 h decreased the dispersion in the interval between AI and ovulation. There was an interaction (P<0.05) between the time of AI and expression of estrus. Pregnancy rates were higher in cows in estrus at 48 h that were AI at that time (47.8%) and those that were not in heat at 48 h and were AI at 60 h (53.4%) than those who were not in estrus at 48 h and were FTAI at that time (19.4%) and those that were in estrus at 48 h and were FTAI at 60 h (29.7%). In Experiment 3 PR was evaluated based on the addition or not of GnRH to cows not showing estrus at 48 h. Pregnancy rate was higher (P<0.05) in cows that were not in estrus at 48 h, that received GnRH at that time and were AI at 60 h than in cows not showing estrus and 48 h that were also AI at 60 h but without GnRH (56.0% vs 39.8%). Experiment 4 evaluated factors that may affect PR in cows treated with the protocol developed in this experiment. It was found that the heat stress of January, a body condition score <2.75 and the presence of only small follicles (<10 mm and without CL) adversely affect PR. In conclusion, the results of this series of experiments confirm the hypothesis that the inclusion of a dose of GnRH and the modification of the time of AI increase PR in lactating dairy cows treated with P 4 devices and estradiol that does not express estrus at the time of FTAI.
The primary objective of implementing embryo transfer in meat operations is to accelerate the rate of genetic progress in the herd. Among the main factors that affect the use of these technologies are related to the nutrition, the management and the synchronization of the estrus. As a result of research conducted in the last 20 years, the use of the receptor has increased through the application of protocols that synchronize ovulation and allow the transfer of embryos without the need for oestrus detection, generally known as fixed-time embryo transfer. (FTET). Although these protocols have functioned well for several years, attention has recently been directed to the effect of estrus expression and estradiol concentrations during pre-ovulatory follicle growth on embryonic growth and pregnancy. The experiments reviewed in this document demonstrate that oestrus expression is positively associated with high pregnancy rates and reduced pregnancy losses in recipients receiving frozen / thawed bovine embryos produced in vitro and in vivo.
Sperm morphology evaluation is an important parameter for determining the quality of semen and predicting fertility in rams. Different staining methods have been developed to detect the morphological status of sperm, but there is no optimized protocol, especially for animals yet. This study was designed to compare the results using SpermBlue®, Diff-Quick, and Coomassie Blue stain in the morphological evaluation of epididymal ram semen. In the study, samples collected from a Bafra ram (epididymal sperm/ram) known to have a good breeding history were diluted with Trisbased diluent and frozen. After thawing for each straw, three semen smears were made and stained with SpermBlue®, Diff-Quick, and Coomassie Blue. All morphological parameters were evaluated using a light microscope. 100 spermatozoa were examined randomly and classified according to their characteristics for each slide. In identifying morphological abnormalities, the staining protocols have compared amongst themselves, and no significant difference between DiffQuick and SpermBlue® staining methods was observed. However, significant differences were observed in midpiece abnormalities when SpermBlue® and Coomassie-Blue staining methods were compared, while significant difference was found in total abnormality in SpermBlue® and DiffQuick staining comparison (P <0.05). As a result, all staining methods evaluated can be easily optimized for laboratory conditions and used in the morphological analysis of ram semen.
For years, researchers have been seeking to manipulate sex before conception. Although the use of sexed semen has been of great interest in the past 20 years to inseminate dairy heifers, the massive use of sexed semen was somewhat limited because fertility was compromised and could not be solved by increasing the number of sperm per inseminating dose. However, new simplified and less traumatic procedures for sperm have recently been developed that have significantly improved pregnancy rates obtained with sexed semen. However, for the massive use of this technology it is necessary to develop fixed-time Artificial Insemination (FTAI) programs for sexed semen. Therefore, a series of experiments were carried out on heifers in cows with calf on foot. In all cases the animals were synchronized with the help of paint at the base of the tail to detect the presentation of jealousy and optimize the IATF schedules. Although pregnancy rates were generally higher for conventional semen than for sexed semen, pregnancy rates with sexed semen were 45 to 50%. Therefore, the results of the experiments carried out show that IATF protocols can be adapted for the use of sexed semen and thus enable the massive use of this technology.
Cows that do not become pregnant during early lactation after successive inseminations are usually classified as repeat-breeder cows. Furthermore, heat stress has a pronounced deleterious effect on fertility in dairy herds throughout the world, especially in hot and humid summer months in tropical and subtropical areas. Studies describes the detrimental effect of heat stress on reproduction, with emphasis on carry over effects of heat stress on oocyte quality, embryo development and P/AI. Additionally, lower fertilization rate is also an important characteristic associated with repeatbreeder cows. In repeat-breeder cows, decreased fertility has been related to a deleterious effect on oocyte quality. Thus, the fertility problems observed in repeat-breeder and heat stress cows may be related to poor oocyte quality associated with a metabolic status. Studies support thar embryo transfer can be a practical and effective solution to achieve pregnancy in repeat-breeder and heatstressed dairy cows (with reduced oocyte quality and fertility to insemination).
It is well established that the timing of the first pregnancy in heifers has a major influence on their lifetime reproductive performance. However, zebuine heifers typically show delayed puberty with the first calving often occurring at around 36–48 months of age. Several factors affect the reproductive efficiency in heifers, such as weight, age, body condition, uterine development and genetics. The thickness of subcutaneous fat efficiently predicts the risk for the establishment of puberty and pregnancy in 14-month-old Nelore heifers submitted to FTAI. It was found that heifers with greater subcutaneous fat thickness were more likely to achieve puberty and to become pregnant at FTAI. Through adequate nutritional management combined with genetic selection for sexual precocity, it is possible to obtain also satisfactory reproductive efficiency in precocious primiparous. When the basic requirements (mainly nutrition and genetics) are met, the anticipation of the conception of heifers can be successfully established, improving the productivity and profitability of breeding livestock.
The objective of this work was to carry out a review of how reproductive efficiency can be improved in dairies. Dairying in the world is demanding changes to which we have to constantly adapt if we want to remain in the system. The constant variation in the price of a liter of milk paid to the producer and in the cost of inputs that are affected by the demand and the economic policies of each country, have installed in the dairy producer the idea that milk production is a bad business due to the low profitability of the sector with respect to other less demanding agricultural activities such as agriculture. However, when analyzing production costs, there is a very wide variation between farms depending on the efficiency and effectiveness with which each establishment performs. The dairy chain has historically presented a dynamic conditioned by demand (internal and external) that ends up setting prices for the primary producer, thus conditioning the productive and technological path of the sector, giving rise to recurring conflicts of interest between producers and industrialists. The subsidy policies that are applied many times end up solving the specific situation of the producer, complicating the situation of the markets of other countries. The countries with the largest surpluses of milk, such as New Zealand, the United States of America, Germany, France, Australia and Ireland, have to sell a large part of their production to other markets, often complicating the producers of the importing countries, for which they must become increasingly efficient in order to compete with countries that subsidize primary production.
Fixed–time artificial insemination (FTAI) in cattle has had a marked growth in South American countries in the last two decades, mainly based in the use of pharmacological treatments with estradiol and progesterone. To improve the pregnancy rate obtained with this technology, the focus has recently been on other aspects of the hormonal protocols that had not been considered previously. The most novel approaches propose studying preovulatory strategies to improve postovulatory conditions to improve pregnancy outcomes. In this paper we review the main results obtained by extending the proestrus length (i.e., the interval from progesterone removal to insemination). The results show that these new approaches allow an improvement in pregnancy rate, which deserves to be taken into account to be included in the implementation of FTAI programs.
The objective was to evaluate the maturation time to obtain oocytes in Metaphase II (MII) stages and the rate of division and blastocysts after fertilization in alpacas. For this, the ovaries were obtained from benefited animals. Experiment 1: 441 oocytes were distributed in four times (26, 32, 38 and 42 hours) and matured in TCM-199. At the end of the maturation time, the oocytes were fixed in an ethanol solution: acetic acid (3:1) and stained with aceto-orcein (1%). Experiment 2: 959 oocytes were cultured under the same conditions as experiment 1 and fertilized with sperm obtained by artificial vagina and selected by the Swim Up method. At the end of the fertilization time, the presumed zygotes were cultured in SOFaa medium for 7 days. In experiment 1, 39.6 ± 8.0, 49.1 ± 18.6, 45.9 ± 9.1 and 38.2 ± 9.9% of oocytes were obtained in Metaphase-II for 26, 32, 38 and 42 h of maturation, respectively, without statistical difference (p>0.05). In experiment 2, the cleavage rate was 45.7 ± 3.5, 51.3 ± 10.2, 52.8 ± 5.6 and 50.9 ± 3.1 with no statistical difference between treatments (P>0.05) and the blastocyst rate was 20.5 ± 3.4, 30.5 ± 5.8, 21.2 ± 2.5 and 22.8 ± 3.5 for 26, 32, 38 and 42 hours, respectively, with a statistical difference between 32 hours and 26, 38 and 42 hours (p<0.05). The results obtained suggest that 32 hours of maturation are required to obtain 30.5% of blastocysts
This study evaluated the effect of oxygen supplementation on the hatchability of fertile eggs of the Creole hen at 2718 meter above sea level. The experiment was carried out with 1408 hatching eggs obtained at age different of the breeders (25, 30, 34 and 38 wks.). Eggs were assigned to four incubators with different periods of oxygen supplementation: treatment without oxygen supplementation (SS) and oxygen supplementations at week 1, week 3, and weeks 1 and 3. Body weight, hematocrit, and hemoglobin were determined of embryos of 18 days incubation and newborn chicks. Hatching percentage was calculated based on the number of fertile eggs. Early and late embryonic mortality was determined. From the fertile eggs supplemented with oxygen during the first and third weeks, a higher percentage of hatched chicks (82.60%) and lower embryonic mortality were obtained. In the group SS, a low percentage of hatched chicks (28.32%) and high embryonic mortality were found. Oxygen supplementation to fertile creole hen eggs under hypobaric conditions in the Cajamarca valley did not increase hemoglobin concentration or improve body weight of hatched chicken.
The feeding program carried out during the pre-partum and post-partum period in dairy cows is crucial to preserve their reproductive performance. The goal of the present study was to assess the effect of black wattle (Acacia mearnsii) supplementation on ovarian activity, uterine involution, and hormone profiles in dairy cows maintained at high-altitude conditions. A total of 10 Holstein dairy cows were divided into 2 groups: control (C; n=5) and supplemented group (AM; n=5). A dehydrated black wattle (AM; Acacia mearnsii) supplement was administered to AM group and adjusted to 22% crude protein per animal (Dehydrated Supplement; MS= 2.7 kg AM/day per cow, ~20% of total DM). Body weight (BW) was recorded individually in different periods (day 15 pre-partum, and day 19, 34, and 49 post-partum). Ovarian [follicles (FL), corpora lutea (CL)] and ovarian diameter (OD; mm)] and uterine structures [uterine horn thickness (HT; mm), cervix length (CrL; cm) and diameter (CD; cm), and uterine involution %] were assessed till day 34 postpartum. Moreover, hormone profiles such as thyroid-stimulating hormone (TSH; IU/mL), follicle-stimulating hormone (FSH; IU/mL), luteinizing hormone (LH; IU/mL), and progesterone (P4; ng/mL) were measured till day 49. Statistical differences were observed in BW when day 15 prepartum was compared to day 19 postpartum (p=0.05). However, no differences were observed between C and AM group regarding BW within each timepoint (p>0.05). No changes were observed in FL over time irrespective of the group studied (p<0.05). However, FL was significantly different when C and AM group were compared within each time-point (p< 0.05). Although no significant differences were observed on day 5 post- partum regarding the number of CL, significant differences were observed on day 25 and day 34 postpartum between groups (p> 0.05). OD increased from day 5 to Day 35 post-partum irrespective of the group assessed and differ between groups within time-points (p< 0.05). Differences were observed in TSH within day 19 and day 49 post-partum (p< 0.05). FSH was statistically different between groups on day 19 post-partum (p< 0.05). LH was more variable than TSH and FSH among and within time-points and groups (p< 0.05). Differences were observed between groups regarding P4 within day 19 and 49 post-partum (p< 0.05). In conclusion, overall although no differences were observed in BW between groups or time-points, the supplementation with AM showed differential patterns in reproductive tract structures/dimensions and hormone levels in dairy cows maintained at high-altitude conditions
Glycerol (Gly) is the cryoprotective agent (CPA) most widely used to cryopreserve rooster semen. However, its cryoprotection, toxicity, and efficacy may vary in different breeds of roosters (e.g.,fighting rooster). In this sense, this investigation evaluated the effect of different concentrations of Gly added to the Lake-Ravie extender on the kinetic variables and plasma membrane integrity (PMI, equivalent to viability) of rooster spermatozoa. A total of 42 semen ejaculates from 6 Spanish fighting roosters (collected in 7 weekly sessions) by dorsal massage technique were used to conform 7-pools. Each pool was divided into 6-aliquots and then 6 treatments were formed according to Gly concentrations: 0 (control), 2% (Gly-2), 4% (Gly-4), 6% (Gly-6), 8% (Gly-8), and 10% (Gly10). The samples were frozen in static liquid nitrogen vapors, and their post-thaw sperm quality was analyzed using the CASA system (SCA-2018®) and Fluorescence (PI). Results after thawing showed that total (MT, %) and progressive (MP, %) motilities were higher (P<0.01) with the Gly-8 treatment compared to the control group and the Gly-4 and Gly-6 treatments. Regarding the postthaw kinetics, the oscillation (WOB, %) and the beat-cross frequency (BCF, Hz) were higher (P < 0.05) in the Gly-6, Gly-8, and Gly-10 treatments compared to their control. Finally, the PMI (%) was greater with the Gly-8 treatment compared to the Gly-2 treatment (P<0.05) and the control group (P<0.01). In conclusion, the addition of 8% glycerol to the freezing medium produced better sperm cryosurvival based on higher kinetics and integrity of the plasma membrane of fighting rooster spermatozoa.
Equine chorionic gonatrophin (eCG) is a high molecular weight glycoprotein produced by endometrial cups in the mare. ECG has primarily LH activity in the mare, but has FSH or LH activity in the cow, depending on the recipient populations in the ovary. Although eCG has been used to induce superovulation in a variety of species, several studies have demonstrated the beneficial effects of eCG on fixed-time AI (IATF) programs in beef and dairy cattle, and embryo recipients. Under these circumstances, eCG has stimulated the development of the ovulatory dominant follicle and produced ovulation of a larger follicle, resulting in a larger and more functional LC and longer embryo survival. In suckling beef cows, 400 IU of eCG at the time of removal of a progesterone device resulted in increased conception rates after IATF,and in dairy cattle managed in pastoral systems, the eCG administration resulted in a significant increase in pregnancy rates, especially in cows in anestrus. In embryo recipients, treatment with eCG has resulted in increased pregnancy rates after embryo transfer. This presentation will review some of this data and explore strategies to use the potential benefits of eCG to improve bovine reproduction.
The objective of this study was to determine the effect of glycerol (GL), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) at temperatures of 5 °C and 16 °C of equilibrium on sperm quality, after cryopreservation. 6 males were used in the semen collection, motility parameters were evaluated with the CASA system, viability, acrosomal integrity and mitochondrial activity by flow cytometry in fresh and thawed semen. We collected 30 ejaculates of which 10 were discarded for not fulfilling (volume ≥ 1 ml and total motility ≥ 60 %), the 20 ejaculates were mesclo in a tube to form pool carrying out the assessment of the initial quality of fresh semen, obtaining a total motility (MT) 61,6 ± 3,4%, progressive motility (MP) 51,7 ± 3,7%, viability 78,1 ± 9.1%, mitochondrial activity (MA) 77,4 ± 7,7 and acrosomal integrity 88,9 ± 11,5%. After cryopreservation no equilibrium temperature effect was observed, independently of cryoprotectant in the parameters of seminal quality. Cryoprotective effect (P≤ 0.05) was observed, presenting superior values in the sperm conserved in GL with respect to the rest of cryoprotectants, with significant differences (P ≤ 0,05) in MT, MP, viability and AM and without significant differences in acrosomal integrity. The equilibrium temperature of 16 °C was effective for cryopreservation of alpaca sperm. In addition, the 5% GL preserved better the functional characteristics of sperm compared to 7% (DMF and DMSO).
The objective of this work was to carry out a bibliographic search of all the programs with prolonged proestrus in the fertility of bovine cattle dedicated to meat production. The use of artificial insemination at fixed time (IATF) in cattle has not stopped growing in the countries of the region during the last 20 years, demonstrating that it is a technology that is here to stay. Its adoption curve is in a stage of maturity, having first been adopted by the most innovative and today it is already used by more pragmatic or conservative producers. Therefore, in this review we will emphasize the protocols that we have worked the most in recent years and with which high pregnancy rates have been achieved.
This document is about gene editing mechanisms in animals to enhance certain specific characters in subsequent generations. Gene generation by gene editing is the current revolution in biology. Today it is possible to edit the genome using the CRISPR-Cas tool that allows you to cut and paste very precise DNA sequences like never before. Thus we can improve practically all those characteristics that are determined by a gene, and then completely modify the phenotype of any species.
