Use Of Artificial Insemination Research Articles

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Comparative Study on the Use of Artificial Insemination in Sows in Three Production Units in the Region of Tlachichuca Puebla, México

The main objective of this study was to make a comparison of the use of artificial insemination in sows from three production units belonging to the Tlachichuca-Puebla region, Mexico. These pork production units belonged to the communities of José María Morelos, San Francisco Independencia and Tlachichuca, Puebla respectively. 20 sows were used, 15 of which were primal and 5 multiparous, which were inseminated over a period of 10 months. 20 doses of semen were acquired from the Coyametl Genetic Transfer Center, located in the City of Acatzingo, Puebla. The information obtained from the project was concentrated in the different stages of the study, and stored in an Excel file; the information was processed through the SPSS 10 package for Windows, applying descriptive statistics. 75% of Primal or Nulliparous sows were found which were artificially inseminated and 25% corresponded to Multiparous; 7% of the producers expressed no interest in planning but rather a greater number of kilos when selling, 15% responded interest in the number of offspring and fewer deaths at birth with better feeding of the bellies before and after the births, 78% mentioned the importance of planning a procedure to improve their productivity and reproductive life in their pork production units (PPUs). Regarding litter size, results were obtained through AI in the sows of the 3 UPPs (José María Morelos, San Francisco Independencia and Tlachichuca), a total of piglets in the primals of 163 of the 15 inseminated sows, with an average of 10.8 piglets/sow; In the case of multiparous sows, there were 58 piglets in total with an average of 11.6 piglets/sow. In conclusion, comparative studies are useful for analysis in pork production units, since they are pillars in decision making; as it was, in the reproductive indicators where AI was applied, giving objectivity and greater certainty as was the case in the three communities of the Tlachichuca region, Puebla, Mexico.

First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni.

The Louisiana pinesnake (Pituophis ruthveni) is considered one of the rarest snakes in North America and was federally listed under the Endangered Species Act in 2018. Captive breeding and reintroduction of zoo-bred hatchlings has been successful, however, limited founders in the captive population and the inability to bring new, wild genes into the captive colony presents a major concern for the conservation of this species. The use of artificial insemination (AI) was first applied to snakes in the 1980s but further development of the technique has since received little attention. Our goal was to develop a method of AI for use in breeding Louisiana pinesnakes to facilitate gene flow from wild to captive populations. We inseminated two captive Louisiana pinesnakes with semen collected from one donor male, novel to both females. Timing of AI occurred following the emergence of females from brumation, and when large, distinct follicles were detected using digital palpation. Females were inseminated four and five times over a period of 14 and 19 days, respectively, using fresh and 2-day refrigerator stored semen. One female laid seven eggs, which resulted in four fertile eggs and two viable hatchlings, while the second female produced three fertile of seven eggs laid but no viable hatchlings. Genetic analyses confirmed the donor male was the sire of hatchlings. This is the first successful AI of an endangered snake species and provides a framework for the use and optimization of assisted reproductive technologies for use in conservation breeding programs.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Comparative characteristics of protocols for cryopreservation of ram semen

Purpose: study and comparison of protocols for cryopreservation of sperm of stud rams.Every day the popularity of sheep farming in Russia is increasing, since sheep are unpretentious in maintenance and at the same time they are multiproduct animals, providing the population with the necessary raw materials (wool, felt) and food products (meat, milk). For this reason, artificial insemination is of fundamental importance in the development of this branch of livestock farming, an important part of which is the correct cryopreservation of ram sperm. There are quite a few advantages of using cryopreserved sperm. Cryopreservation of sheep sperm contributes to the spread of the use of artificial insemination in this branch of the livestock industry. Freezing technology largely ensures the preservation of genetic material and its transportation, eliminating geographical barriers in the use of artificial insemination, and allows increasing the load on the ram sire without risk to his reproductive health. However, during the process of cryopreservation, sheep sperm receives irreversible damage due to cold shock, osmotic stress and oxidative processes, which lead to a decrease in the ability to fertilize sperm. Thus, chromatin may be damaged, membrane permeability increased, hyperoxidation and the formation of reactive oxygen species are possible, which affects the ability to fertilize. As a result, frozen ram semen may have low fertility. For this reason, it is extremely important to select an up-to-date freezing and thawing protocol with maximum recovery of viable and functional sperm of a given animal species for successful further use in artificial insemination. Technological advances in various fields of biotechnology have led to improvements in protocols, methods and equipment used in the laboratory, which have a positive impact on the reliability, accuracy of the procedure. This article summarizes research on the effects of different cryopreservation procedures, cooling rates, cooling times and thawing protocols on ram semen quality. In addition, a correct protocol for cryopreservation of Romanov breed sperm was formed, based on the results of our own research.

PSXIV-4 Body Condition and Production Status Impacts on Estrus Indicators in Beef Cattle on a Producer Farm

Abstract The use of artificial insemination (AI) in cattle can increase farm profitability by increasing the genetic merit of offspring, but animal body condition (fatness) and stage of production can influence breeding efficacy. Therefore, during an on-farm AI session, the effect of body condition score and production status (nursing offspring or not) on indicators of estrus (readiness for breeding) was examined. Thirty-six cows 3 to 12 years of age and having previously had at least one calf were used. Cows were estrus synchronized by the owners using the Co-Synch with CIDR fixed time AI protocol (GnRH and CIDR on day 0 followed by CIDR removal and prostaglandin on day 7 with AI and GnRH on day 10). At AI using cryopreserved semen from a commercial source (Premier Select Sires), body condition score was assessed (1-9; 1 = emaciated, 9 = obese), and indicators of estrus (presence of mucous, pink color of vulva, turgid and open cervix and easy insemination) were noted. On this farm, body condition scores for the animals the producer chose to estrus synchronize ranged from 3 to 5. Animals were nursing calves 3 to 5 months of age (n = 15) or were not nursing calves (n = 21). Statistical analysis (with SAS) indicated that nursing negatively impacted body condition as well as number of estrus indicators noted at AI and that body condition tended to be positively related to the number of estrus indicators noted.

103 In Utero Exposure to Maternal Heat Stress Impairs Leydig Cell Development and Function in the Boar

Abstract Due to the nearly exclusive use of artificial insemination, a typical U.S. boar sires ~8,398 progeny per year which is a 99.7% greater gene transmission rate compared with a sow (~23.1 progeny/year). Therefore, the boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, boar fertility is threatened by climate change. While boar studs are cooled to prevent heat stress, it is cost prohibitive to efficiently cool the six million pregnant sows that are exposed annually, leading to in utero heat stress (IUHS) of developing progeny. The IUHS of the boar reduces the number of pre-spermatogonia in neonatal testes and decimates subsequent semen quality by reducing both total sperm count (24% decrease) and increasing the proportion of abnormal sperm (42% increase). These data suggest that IUHS programs a lifetime of testis dysfunction reducing the breeding potential of boars. However, the biological mechanisms mediating this effect are undefined. The objective of this study was to determine how IUHS affects Leydig cell development and the testicular proteome. Pregnant sows were exposed to either heat stress (28 to 38°C; n = 18) or thermal neutral (17 to 22 °C; n = 18) conditions from day 27 to 57 of gestation. Neonatal boars exposed to IUHS (n = 23) or in utero thermal neutral (IUTN; n = 29) conditions were castrated at 3 days of age. Testes were preserved for histological analysis and proteomics. Leydig cells within IUHS testes were hypertrophic compared with IUTN controls (147.3 ± 1.6 µm2 versus 132.8 ± 1.4 µm2; P < 0.0001). Moreover, IUHS decreased the number of Leydig cells (Per microscopy field) compared with IUTN males (72.7 ± 2.0 Leydig cells/field versus 93.5 ± 1.8 Leydig cells/field). The testicular proteome was interrogated in a subset of animals (n = 8/treatment). A total of 519 proteins (32% of those detected) were differentially abundant between IUHS and IUTN testes (278 proteins down-regulated, 241 proteins up-regulated) including proteins important for Sertoli (e.g., insulin-like growth factor binding protein), Leydig (e.g., CYP19), and germ (e.g., glyceraldehyde 3-phosphate dehydrogenase) cell function. Notably, gene ontology analysis revealed that both steroid & cholesterol biosynthesis pathways were enriched (P < 0.0001) indicating differential expression of proteins needed for testicular steroidogenesis. Notably, CYP19 (enzyme expressed in porcine Leydig cells that converts testosterone to 17β-estradiol) was down-regulated in IUHS testes. Therefore, these data suggest that IUHS leads to Leydig cell dysfunction in boars. The importance of Leydig cells to male reproduction is unequivocal; thus, IUHS represents a mounting threat to male fertility, especially in the face of climate change.

RISK-ORIENTED PREDICTION OF PREGNANCY LOSS PATHOLOGY BASED ON A PERSONALIZED APPROACH

The increase in the frequency of pregnancy loss and the negative prognostic trend with the probable development of the demographic crisis in connection with the martial law in Ukraine and the mass migration of the female population provoked by it, requires the search for all possible ways of prevention and prophylactic of this pathology.
 Aim. Development and scientific substantiation of a risk-oriented model of prevention of pregnancy loss.
 Material and Methods. In the course of creating a model for determining the probability of the appearance of symptoms of pregnancy loss, we used a step-by-step method of logistic regression with the gradual exclusion of unreliable signs according to the Wald method.
 Results and Discussion. In the process of sociological research, the importance of 30 risk factors was analyzed, and 14 factors were singled out from among them using the logistic regression method, which, when combined, have a probable impact on the development of pregnancy loss. Namely: emotional strain/stress in everyday life, first ultrasound and biochemical screening, history of herpes type 1, chlamydial infection, COVID-19, tonsillitis, allergic rhinitis/sinusitis, autoimmune thyroiditis, presence of type I diabetes, use of artificial insemination, wine consumption, additional use of folic acid and drugs, marital status, and computer work. Among these 14 factors, three have a preventive effect, while the other 11 increase the risk of developing the pathology of pregnancy loss. This preventive model is reliable (p<0.001) and was transformed into the application "Individual prediction of the occurrence of pregnancy loss pathology" created for the Android platform, which provides 95.5% accuracy of the prediction of the occurrence of pregnancy loss pathology.
 Conclusions. The personalized preventive model can be applied by general practitioners/family doctors and obstetrician-gynecologists in order to form risk groups among pregnant women and prevent the occurrence of pregnancy loss pathology.

Three decades of progress in artificial insemination in rabbit farming: a review

The commercial use of artificial insemination (AI) in rabbit farming is relatively recent, especially when compared to other species such as cattle or swine, in which AI has been used for more than 60 years. The large-scale use of AI in rabbit farming dates back to the late 80s. However, despite its short journey, it has not stopped evolving. Although there have been numerous changes, in this review article we aim to highlight two important milestones in optimisation of this technique: the introduction of biostimulation and the addition of Gonadotropin-releasing hormone (GnRH) analogues to the seminal dose to induce ovulation. In the former case, by means of different methods of biostimulation, such as feed and light flushing and/or separation of the litter in the days prior to AI, the use of hormones to synchronise heat with the moment of AI was practically eliminated. Nowadays, the possibility of using pheromones with the same objective is under research, even to increase ovulation rate or improve semen production. Although there are pheromones on the market labelled for use in other species, in the case of rabbit the knowledge of them is limited. Nevertheless, given the verified effects that pheromones produce in other animals, expectations are high. In the latter case, after several attempts by using other methods, the technique commonly used to induce ovulation was the intramuscular administration of GnRH or its synthetic analogues. However, in recent years, it has been proven that administration of GnRH through the vagina is possible, added to the seminal dose, which offers numerous advantages regarding health, animal welfare and the workforce needed. Recently, the European Medicines Agency (EMA) approved this practice, so in the near future it will probably become the most widely used method. Even so, there is still room for improvement, as the dosage of GnRH needed is higher than the one administered intramuscularly. Research on this topic allows us to predict that this problem should be solved in the coming years. Other alternatives such as the β-Nerve Growth Factor need further research to become a feasible option.

The importance of historically popular sires on the accuracy of genomic predictions of young animals in the US Holstein population

The dairy industry is known for its extensive use of artificial insemination, which has resulted in a population where most animals can be traced back to only a few sires. Due to their relatedness to the population, old influential sires could still contribute to the accuracy of genomic predictions. The objective of the study was to identify the impact of historically influential sires on the recent population. This was tested by constructing a genomic relationship matrix using recursion with different sets of sires. Differences in prediction accuracies with different sets are indicative of how important each set is. Recursion coefficients linking young animals to those sets reveal the relative importance of specific sires to the prediction accuracy of recent animals. The data included ∼10 million scores for stature and fore udder attachment (FUA) measured from 1983. Genotypes of 569,404 animals were available. Sire sets included the 100 most popular sires born within different time periods. Computations were with single-step genomic BLUP. In general, the younger sires had higher prediction accuracies than the oldest sires, even though they generally have fewer progeny. The accuracy of evaluation for stature was increased from 0.54 with the most popular sires born before 1981 to 0.69 with sires born from 2001 to 2010, while the accuracy for FUA increased from 0.47 to 0.61. The accuracy achieved using the overall 100 most used sires was 0.66 for stature and 0.58 for FUA. All 100 sires from each period were combined in a subset to determine the importance of each sire relative to all 400 animals in the combined subset. The highest relative impact of a sire that was born within the different time sets was 1.97 for Valiant (before 1981), 1.94 for Blackstar (1981 to 1990), 4.38 for Shottle (1991 to 2000), and 3.09 for Planet (2001 to 2010). The 3 sires among the 400 with the greatest impact were Shottle, Goldwyn (3.73), and Planet. The relative impact of a sire was not strongly related to the number of progeny. For instance, the relative impact of Durham with 34K progeny was 2.29, whereas the impact of O Man with 15K progeny was 3.13. The impact of a sire is also influenced by whether it was used as a sire of sires. Results show that younger sires are more relevant to the accuracy of breeding value prediction in the recent population.

Compensability of an enhanced incidence of spermatozoa with cytoplasmic droplets in boar semen for use in artificial insemination: a single cell approach

This single cell study aimed to clarify whether an elevated incidence of sperm with a retained cytoplasmic droplet (CD) can be compensated by a higher sperm number in boar semen doses to maintain fertility. Cluster analysis of motile spermatozoa (ten boars) revealed that spermatozoa with a CD are underrepresented in the fast, linearly moving sperm cohort compared to morphologically normal sperm. Nonetheless, the response to the motility stimulator procaine was barely affected in spermatozoa with distal CD (Cramer’s V = 0.14), but moderately affected in sperm with proximal CD (V = 0.28). Viability was lower in sperm with distal CD (p < 0.05) but not with proximal CD compared to normal sperm during 168 h storage of extended semen samples (n = 11) and subsequent thermic stress. Morphologically normal sperm from normospermic samples (n = 10) or samples with a high incidence (≥ 15%) of sperm with CD (n = 9) had similar motility patterns and responses to procaine. The origin of morphologically normal sperm had no effect on sperm viability (p > 0.05; n = 26). In conclusion, a moderately enhanced prevalence of sperm with CD seems to be compensable by an increase in sperm numbers in boar semen doses.

Use of artificial insemination in dairy herds in Northwestern Rio-grandense, Brazil

Use of artificial insemination in dairy herds in Northwestern Rio-grandense, Brazil

The quality and fertilizing ability of the ram semen cryopreserved around 40 years ago.

The aim of the study was to verify the quality of ram semen, frozen in 1982-1983, from the historical collection of the Bank of Biological Material of the National Research Institute of Animal Production. A total of 18 ejaculates from 3 Świniarka type rams were analyzed to assess sperm motility (subjectively), total motility, progressive motility, sperm concentration (CASA), membrane integrity (SYBR-14/PI) and chromatin structure (SCSA). In order to determine sperm fertilizing ability 49 ewes were intracervically inseminated (200×106 sperm per AI) with frozen- thawed semen 12 and 24 hours after detection of estrus. Sperm motility parameters, membrane intact spermatozoa and DFI did not differ among the analyzed rams. Spermatozoa concentration was significantly higher for ram no. 2 than for rams no. 1 and 3. The lambing rates (27.3 to 36.0%) did not differ significantly for individual rams. The ram semen, which had been stored for around 40 years, showed satisfactory quality and fertilizing capacity, allowing for its use in artificial insemination.

Use of a cow-side oestrus detection test for fertility management in Kenyan smallholder dairy herds.

Background: The use of artificial insemination (AI) has great potential to improve smallholder dairy herds in Africa, however poor success and, in some situations, high costs in Kenya, have been discouraging. Effective AI requires accurate oestrus detection and the measurement of progesterone (P4) can be used to indicate oestrus as well as non-pregnancy. A cow-side progesterone lateral flow test, P4 Rapid, was evaluated as an aid to detect oestrus and non-pregnancy in Kenyan dairy cows, and assessed for association with AI efficiency. Methods: A total of 527 cows were enrolled in the study, from two counties in central and southern Kenya. Cattle in the test group (n = 308) were presented when suspected to be in oestrus and tested with the P4 Rapid (low P4 = oestrus, medium P4 = inconclusive, high P4 = not in oestrus/pregnant). Cattle with low P4 were inseminated. Cattle in the control group (n = 219) were inseminated when oestrus behaviour was detected i.e. standard practice. Results: Of the total P4 Rapid tests performed (n = 745), 1.5% were inconclusive, with the true accuracy of the test between 87-97%. Conception rates were not significantly higher in the test group (83.9%) compared to the control group (77.9%). Abortion rates were not significantly different between the control (9.5%) and test groups (8.2%). In the test group, 6.2% (19/308) cows showed a medium or high P4 level on day 0 and nine of these were subsequently found to have been already pregnant. Conclusions: The data indicated that the P4 Rapid test can be a useful tool to assist farmer decision-making in the confirmation of correct timing for AI, and importantly may avoid unnecessary inseminations in pregnant animals, thus reducing the risk of AI-induced abortion.

The vaginal and uterine blood flow changes during the ovsynch program and its impact on the pregnancy rates in Holstein dairy cows

AimOvSynch is a hormonal protocol for synchronization of estrus and use of artificial insemination (AI) at an optimal time without adverse effects on the ovaries or uterus. This study investigated the use of noninvasive color Doppler ultrasound to assess changes in uterine and vaginal blood flow during the Ovsynch program for synchronization of estrus and its relation to the pregnancy rates in Holstein cows.Materials and methodsThe experimental cows received an intramuscular dose of 10 μg of a GnRH analogue (G1), followed 7 days later with an intramuscular injection of synthetic prostaglandin F2α (P: PGF2α) analogue (500 μg cloprostenol sodium), and given a 10 μg, injection of the GnRH analogue (G2) i.m. 48 h after the PGF2α treatment, and the cows were bred 14-16 h after. Uterine and vaginal perfusion were investigated by performing transrectal Doppler ultrasonography of both the uterine and vaginal arteries in Holstein cows at different time points during the Ovsynch program to determine: peak systolic velocity (PSV), time-averaged maximum velocity (TAMV), the volume of blood flow (BFV), pulsatility index (PI), resistance index (RI), resistance impedance (S/D) and diameters of uterine (UA) and vaginal (VA) arteries. Steroid hormones were also assayed. Transrectal ultrasonography (TUS) was performed at 32 and 60 days to confirm the pregnancy per artificial insemination (P/AI).ResultsThe uterine PSV, TAMV, and PV were greater at the time of the cloprostenol sodium and second GnRH injections (p<0.05) than at the time of the first GnRH injection. The vaginal PSV, PV were greater at the time of the cloprostenol sodium than at the time of the first and second GnRH injections (p<0.05). The receiver operating characteristic curve (ROC curve) indicated a high correlation between the uterine and vaginal blood flow and the rate of the pregnancy (p<0.05). The area under the ROC curve was 0.920 and 0.87 (p<0.05) for vaginal and uterine arteries respectively at time of G2. The serum levels of progesterone, estrogen and cortisol were correlated with the P/AI (p<0.05). The P/AI significantly decreased from 43.9 % at 32 d to 35.37 % at 60 d.ConclusionThese results indicate that noninvasive Doppler ultrasonography is a valid method to evaluate changes in the characteristics of uterine and vaginal blood flow in cows during the Ovsynch protocol. Furthermore, vaginal and uterine blood flow are two determinant factors for the higher conception rates in Holstein dairy cows.

European Regulations on Camel Germplasm Movement within the European Union: A Current Framework Based on Safety.

Simple SummaryDelegated Regulation (EU) 2020/686 (hereafter Reg. 686) regulates the traceability and animal health for the movement of germinal material of camels within the EU. Given that the camel breeding industry is in a strong phase of growth, an amount of germinal material of terrestrial animals—including those belonging to the Camelidae family—is moved between the member states. The authors performed an analysis of Reg. 686 addressing veterinarians and breeders who want to sell high-quality germinal material or genetically improve their herds.With the aim of developing livestock breeding, the Delegated Regulation (EU) 2020/686 (hereafter referred to as Reg. 686) has taken steps to define traceability and animal health for the movement of germ material within the European Union (EU), including that of camelid species. Despite the economic importance of the camel market and the efforts of the EU to regulate their movements, there are considerable difficulties in the collection of semen and its freezing, limiting the use of artificial insemination in this species. If, on the one hand, there is little diffusion of the camel breeding and, consequently, limited diffusion of animals and germplasm, there will probably be a significant increase over the years. To avoid the spread of emerging diseases—or even those no longer present in Europe—the entry of genetic material from non-EU countries must be strictly monitored. Camels are rarely clinically compliant, but can transfer even fatal diseases to domestic ungulate farms in the EU. Based on these considerations, we conducted a narrative review of the European regulations on this issue, focusing on aspects related to their application in camels.

¿ES POSIBLE MEJORAR LA FERTILIDAD CON PROTOCOLOS DE PROESTRO PROLONGADO? BASES FISIOLÓGICAS, RESULTADOS Y NUEVAS PERSPECTIVAS

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.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp) kept in captivity.

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

Use of a cow-side oestrus detection test for fertility management in Kenyan smallholder dairy herds.

Background: The use of artificial insemination (AI) has great potential to improve smallholder dairy herds in Africa, however high costs and often poor success rates have been discouraging.  Effective AI requires accurate oestrus detection and the measurement of progesterone (P4) can be used to indicate oestrus as well as non-pregnancy.  A cow-side progesterone lateral flow test, P4 Rapid, was evaluated as an aid to detect oestrus and non-pregnancy in Kenyan dairy cows, and assessed for association with AI efficiency.  Methods: A total of 527 cows were enrolled in the study, from two counties in central and southern Kenya.  Cattle in the test group (n = 308) were presented when suspected to be in oestrus and tested with the P4 Rapid (low P4 = oestrus, medium P4 = inconclusive, high P4 = not in oestrus/pregnant).  Cattle with low P4 were inseminated.  Cattle in the control group (n = 219) were inseminated when oestrus behaviour was detected i.e. standard practice. Results: Of the total P4 Rapid tests performed (n = 745), 1.5% were inconclusive, with the true accuracy of the test between 87-97%.  Conception rates were not significantly higher in the test group (83.9%) compared to the control group (77.9%). Abortion rates were not significantly different between the control (9.5%) and test groups (8.2%).  In the test group, 6.2% (19/308) cows showed a medium or high P4 level on day 0 and 9 of these were subsequently found to have been already pregnant. Conclusions: The data indicated that the P4 Rapid test can be a useful tool to assist farmer decision-making in the confirmation of correct timing for AI, and importantly may avoid unnecessary inseminations in pregnant animals, thus reducing the risk of AI-induced abortion.