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A direct radioimmunoassay method for the measurement of progesterone in blood dried on filter paper has been developed for the early pregnancy diagnosis in sows, as well as for monitoring progesterone levels during the oestrous cycle. Pregnancy diagnosis was performed with 95 sows on Days 17–22 after artificial insemination (AI). The cut-off value for pregnancy diagnosis of 7.5 ng/ml was calculated (mean±2S.D.) from the progesterone concentrations measured on the same days from non-inseminated animals. There were 85 cases considered pregnant on the basis of progesterone concentration, leaving 10 animals non-pregnant. The accuracy for the positive cases was 98.8%. Two of the 10 sows considered as negative subsequently farrowed, giving an accuracy of 80%. The overall accuracy of the method was 96.8%. The blood-spot assay may be a useful tool for early pregnancy diagnosis in swine, with respect to sampling, simplicity, speed and accuracy.

American Society of Animal Science members, publishing in Journal of Animal Science (JAS), completed research that resulted in understanding the estrous cycle of cattle, which led to the ability to inseminate cattle on a given day with pregnancy rates similar to those achieved by 21-d breeding by a fertile and sound bull. Research published in JAS led to understanding estrus, ovulation, the estrous cycle, and postpartum interval for cattle (1930s through 1960s) and hormonal factors affecting corpus luteum lifespan of cattle (1950s through 1980s). Research during the 1940s to 1960s, using gonadotropins and progesterone to manage the estrous cycle of cattle, established the concepts for estrous synchronization and stimulated commercial research directed at developing cost-effective progestogen estrous synchronization products, leading to commercially available products from 1967 through today (Repromix, melengestrol acetate, Syncro-Mate-B, controlled internal drug release). Prostaglandin F(2alpha) products were approved for estrous synchronization (1970s, 1980s), and GnRH products were approved for use in cattle to treat ovarian follicular cysts (1970s, 1980s). Research published in JAS was essential for understanding the biology of and potential value of both PGF(2alpha) and GnRH and contributed both to new knowledge and scientific bases for future Food and Drug Administration Center for Veterinary Medicine approval of those products. Research during the1980s through 2000s led to understanding ovarian follicular waves and described the timing of follicular recruitment, selection, dominance, and atresia; this research was essential for the ability to effectively manage follicles to achieve success with timed AI. The knowledge gained through research published in JAS resulted in development of the numerous estrous synchronization and breeding management protocols that are cost-effective and meet the breeding management needs of most beef and dairy enterprises.

Technology in human assisted reproduction has certainly shown great advances during the past couple of decades. In Vitro Fertilisation (IVF) is only one of those techniques which are offering infertile couples the possibility of experiencing parenthood. Infertility affects one's life at the very core-feelings of anger, depression and guilt are very commonly experienced by infertile couples. The causes of infertility are many; amongst the most common are blockages of the fallopian tubes, endometriosis, low sperm counts and poor sperm motility and/or morphology. IVF, however, is not the answer to all fertility problems. IVF is mostly suitable in those cases where there are fallopian tube problems or cervical mucus which is hostile to sperm, since in IVF both the passage of the ovum through the fallopian tube and the passage of sperm through the cervix are by-passed. There are various alternatives to IVF, such as Artificial Insemination (AI) and Gamete Intrafallopian Transfer (GIFT). The latest innovative technique, Intracytoplasmic Sperm Injection (ICSI), which in the future might replace traditional IVF involves the injection of sperm directly into the ovum and is hence ideal for those cases with severe male factor infertility. The setback with all the advances that are being witnessed in assisted reproduction is that they also give rise to many ethical questions. Different countries, with different social, legal, religious and moral backgrounds have different opinions regarding the research on embryos, third-party parenting and gamete/embryo freezing. This study describes the main procedures, techniques and equipments that are used in IVF and its alternatives, as well as the benefits and the ethical implications that such techniques give rise to., peer-reviewed

*Karaşahin, Tahir (Aksaray, Yazar ) *Dursun, Şükrü (Aksaray, Yazar ), In this study, the effect of serum paraoxonase-1 (PON-1) activity on superovulation response and embryo yield was evaluated. The study material comprised 50 Holstein cows aged 3–4 years on postpartum day 90–120 with a body condition score of 3–3.25. A progesterone-based estrus synchronization protocol was initially administered to the selected donors. For this purpose, progesterone source was inserted intravaginally (day 0) and gonadotropin-releasing hormone injection was performed (day 6). Seven days after the insertion of progesterone device, follicle-stimulating hormone injections (total dose of 500 µg in decreasing doses for 4 days) were administered for superovulation. On the morning of the ninth day, prostaglandin (PG) F2α was administered, and the progesterone device was removed from the vagina in the evening on the same day. Two days after PGF2α administration, fixed-time artificial insemination was performed in the morning and in the evening. On the day of artificial insemination, blood samples were taken from the donors to determine the serum PON-1 activity. Uterine flushing was performed seven days after insemination. The results revealed that the serum PON-1 activity (mean ± SD, 562.71 ± 140.23 U/l) of the cows that responded to superovulation (donors with total corpus luteum count of ≥3 in both ovaries) was higher than those (389.91 ± 80.51 U/l) that did not (P

The livestock sector has achieved many technological advances, which have resulted in continued improvements in animal production systems and in the reproductive efficiency of herds. The associated use of reproductive biotechnology and genetic improvements combined with adequate sanitary and nutritional management are essential conditions for sustainable intensified animal production and financial autonomy within farms. Timed artificial insemination (TAI) represents one of the strategies with the greatest impact of expansion in providing genetic improvements and increased reproductive efficiency at a decreased cost. Despite the high proportion of cows receiving TAI, this market still exhibits considerable potential for expansion. After a TAI procedure, approximately 40 to 60% of females become pregnant. This result can vary depending on such factors as the hormonal protocol employed, female category, body condition score, ovarian status, farm management and aspects related to bulls and semen. The fertility and genetic quality of the bull plays an important role in the herd because a single bull can influence the entire production system. Another important strategy is the use of sex-sorted semen associated with TAI, primarily when associated with management practices to improve the pregnancy rate. This paper presents a review of the intensification of TAI, supplying practical information regarding the implementation of TAI commercial programs.

Objectives: The 46,XX disorders of sex development (DSD) is a rare genetic cause of male infertility and possible misdiagnosis of this condition has never been reported. We aim to investigate clinical characteristics and laboratory results of infertile males with possibly misdiagnosed 46,XX DSD. Methods: Between January 2008 and December 2017, a retrospective case series study was performed involving sixteen 46,XX DSD males without azoospermia factor (AZF) deletion. Demographics, clinical features, laboratory results and assisted reproductive technology (ART) outcomes of these patients were depicted, and the underlying accurate diagnosis was also discussed. Results: The mean age was 30.06 ± 5.40 years old. Thirteen patients (81.25%) merely obtained secondary school education. Gynaecomastia occurred in one case, and cryptorchidism appeared in two cases. Testicular volumes were equal to 15 mL on two sides in one patient who had severe asthenozoospermia. Thirteen patients (81.25%) had bilateral atrophic testes which were below 5 mL. The majority of patients were observed with elevated levels of gonadotropic hormones and decreased testosterone values. Neither AZF region nor sex-determining region Y gene was absent among all patients. Twelve patients had normal ejaculatory function, whereas four were diagnosed with ejaculatory dysfunction. Eleven patients (68.75%) were diagnosed with azoospermia. Testicular sperm aspiration was performed in six subjects (37.50%). The pathological results showed that Leydig cell hyperplasia with spermatic failure was found in each case, and no sperm was found in testicular tissue. ART with donor sperm was conducted in 15 patients. Live birth was achieved in three cases through artificial insemination by donor and in one case using in-vitro fertilization by donor. Conclusions: Chromosomal analysis rarely yields 46,XX karyotype combined with no deletion of AZF in infertile males. Under this condition, molecular analysis should be conducted to avoid potential misdiagnosis and false interpretation of other findings.

This study aimed to describe the duration of inflammation after intrauterine infusion of polyvinylpyrrolidone-iodine (povidone-iodine, PVP-I), determine the effect of PVP-I infusion on the subsequent fertility, and evaluate the histopathology of the endometrium in dairy cows. In Experiment 1, 120 lactating clinically healthy Holstein-Friesian cows at 5 weeks postpartum (W5) were equally divided into three groups: intrauterine infusion of 2% PVP-I (PVP), saline (SAL), and no treatment (NTX). Endometrial cytology was performed daily from D0 (W5) to D7 to determine the percentage of polymorphonuclear cells (PMN%) in 44 of the 120 cows. All cows received timed artificial insemination at D17. In Experiment 2, 25 cows were randomly classified into sacrifice at 24 hr or 48 hr after 2% PVP-I infusion (PVP24 and PVP48), and 24, 48, 72, or 96 hr after SAL infusion (SAL24; SAL48; SAL72; SAL96), or no treatment (NTX). Histopathology was performed on the uterus of each cow. In Experiment 1, PMN% was greater in PVP (P

Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination (AI) and assisted reproductive technology (ART). However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications.

Considerable progress in reproduction of dairy goats has been made, with advances in reproductive technology accelerating dairy goat production since the 1980s. Reproduction in goats is described as seasonal. The onset and length of the breeding season is dependent on various factors such as breed, climate, physiological stage, male effect, breeding system, and photoperiod. The reproductive physiology of goats was investigated extensively, including hypothalamic and pituitary control of the ovary related to estrus behavior and cyclicity etc. Photoperiodic treatments coupled with the male effect allow hormone-free synchronization of ovulation, but the kidding rate is still less than for hormonal treatments. Different protocols have been developed to meet the needs and expectations of producers; dairy industries are subject to growing demands for year round production. Hormonal treatments for synchronization of estrus and ovulation in combination with artificial insemination (AI) or natural mating facilitate out-of-season breeding and the grouping of the kidding period. The AI with fresh or frozen semen has been increasingly adopted in the intensive production system, this is perhaps the most powerful tool that reproductive physiologists and geneticists have provided the dairy goat industry with for improving reproductive efficiency, genetic progress and genetic materials transportation. One of the most exciting developments in the reproduction of dairy animals is embryo transfer (ET), the so-called second generation reproductive biotechnology following AI. Multiple ovulation and ET (MOET) program in dairy goats combining with estrus synchronization (ES) and AI significantly increase annual genetic improvement by decreasing the generation interval. Based on the advances in reproduction technologies that have been utilized through experiments and investigation, this review will focus on the application of these technologies and how they can be used to promote the dairy goat research and industry development in the future.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

In this study, we aimed to determine risk factors associated with cytological endometritis (CYTO) diagnosed at artificial insemination (AI) in dairy cows. The CYTO risk factors were evaluated based on 1.625 AI-CYTO samples obtained from 873 Holstein-Friesian cows from in total 18 dairy herds in Flanders (Belgium). The endometrial cytology samples were obtained using the cytotape technique, which consisted of adapting a 1.5 cm piece of paper tape to a standardly loaded AI catheter, covered with a double guard sheet. The polymorphonuclear cells' (PMNs) cut-off point for CYTO at AI was set at >= 1%. We constructed multilevel generalized mixed effect models in order to identify the risk factors associated with the presence of CYTO at AI. The CYTO prevalence at AI was 27.8% at the animal level, while the within-herd level prevalence ranged from 10.7 to 39.7%, with an average of 28.1%. Risk factors associated with the occurrence of CYTO were parity >= 2 [odds ratio (OR) = 1.8], days in milk (DIM) at AI >= 124 (OR = 0.4), and warm months of the year [July (OR = 2.9), August (OR = 2.3), and September (OR = 1.4)]. In conclusion, the present study supports that multiparous cows and cows that are inseminated in the summer months have a higher risk to suffer from CYTO at insemination, while the risk for CYTO is lower when the insemination is taking place at >= 124 DIM. (C) 2017 Elsevier Inc. All rights reserved.

Artificial insemination (AI) and embryo transfer (ET) are the most widely used biotechnologies in the world with the goal of increasing genetic gain and improving reproductive efficiency of beef and dairy herds. The protocols for ovulation synchronization for timed AI (TAI) or ET (TET) are tools that allow artificial insemination or transfer of a high number of embryos in a pre-established moment and without the necessity of estrous detection. Currently, 86% of inseminations in Brazil are performed using TAI (13.6 million TAI out of a total of 15.4 million doses of semen marketed in 2018). With the use of TAI, it was possible to verify that the percentage of artificially inseminated females in Brazil went from 5.8% in 2002 to 13.1% in 2018. The ET market also presented considerable growth in the last 20 years. There was an increase of approximately 8 fold in the number of produced embryos, escalating from 50,000 in 1999 to 375,000 in 2017. In this period, there was a significant increase on the in vitro embryo production, which represented 92.1% of embryos produced in Brazil in 2017. Also, in this period, there was an increase on the embryo production of dairy breeds and reduction on the embryo production of zebu breeds in comparison to data from the early 2000’s. TET increases significantly the number of recipients suitable to receive an embryo. After synchronization, 75 to 85% of recipients present a suitable CL for ET without estrous detection. Currently, many synchronization and resynchronization protocols for TAI/TET have been studied to attend different managements, breeds and animal categories, with predictable and satisfactory results. With the intensification of the use of these biotechnologies, it is possible to obtain elevated reproductive efficiency with increase on the genetic gain, which determines greater productivity and economic return for dairy and beef farms. However, the challenge to keep the market growing in the next decade could depend on some factors, such as: increase of the extension services for producers and of the extension training for specialists, improvement of the technological advances to develop more efficient and cost-effective products and practical protocols, increase the integration between universities, research institutes, veterinarians and industries and also, asses market demand for production of animal protein with higher quality, efficiency and environmental and economic sustainability.

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.

This review aims to describe changes in production environment, management tools and technology to alleviate problems seen with the present hyperprolific sow model. Successful parturition in the pig includes the possibility to express adequate maternal behaviour, rapid expulsion of piglets, complete expulsion of placenta, elimination of uterine contamination and debris, neonatal activity and colostrum intake. We focus on management of large litters, including maternal behaviour, ease of parturition, colostrum production, piglet quality parameters and intermittent suckling. There are also some interesting developments in technology to assess colostrum and immune state of the piglet. These developments may be utilized to improve the success rate of reproductive management around farrowing, lactation and after weaning. We also discuss new insights in how to examine the health of the mammary gland, uterus and ovaries of hyperprolific sows. Finally, we assess the latest developments on breeding and technology of hyperprolific sows, including artificial insemination (AI), real-time ultrasound of the genital tract and embryo transfer (ET). We conclude that 1) for the sow to produce sufficient colostrum, both the behavioural and physiological needs of the sow need to be met before and after parturition. Furthermore, 2) new ultrasound and biopsy technology can be effectively applied for accurate diagnosis of inflammatory processes of the udder and uterus and timing of AI regarding ovulation to improve insemination efficiency. Finally, 3) developments in cryopreservation of germ cells and embryos appear promising but lack of valid oocyte collection techniques and nonsurgical ET techniques are a bottleneck to commercial ET. These latest developments in management of parturition and reproductive technology are necessary to cope with the increasing challenges associated with very large litter sizes.

The objective of this study was to evaluate the effects of post artificial insemination (AI) treatment with intravaginal progesterone device (P4 device) on conception rate, synchronization of returning estrus and plasma P4 concentration in Japanese Black cows. Nineteen cows were treated with DIB (1.0 g P4) from Day 12 to 19 (Day 0=day of the first AI), 27 cows were treated with a CIDR (1.9 g P4) from Day 12 to 19, and 33 cows were not treated after the first AI (control). Estrous behavior was daily examined between Day 20 and 25, and cows returning to estrus were inseminated (the second AI). On Day 19, plasma P4 concentration was not different among DIB, CIDR and control groups. There was no significant difference in conception rate after the first AI among three groups (DIB: 63.2%, CIDR: 66.7% and control: 72.7%). In non-pregnant cows, there was no significant difference in the proportion of cows showed returning estrus between Day 20 and 25 (DIB: 57.1%, CIDR: 22.2% and control: 44.4%), and day of returning estrus was not synchronized. The overall conception rate after the first and second AI was not different among the groups. In conclusion, post-AI treatment with intravaginal devices containing 1.0 and 1.9 g P4 from Day 12 to 19 neither increased plasma P4 concentration nor improved fertility and synchronization of the returning estrus in Japanese Black cows.

This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P

A number of reproductive biotechnologies are currently available to multiply offspring from high genetic merit animals to enhance reproductive efficiency and profitability both in dairy and beef herds. Some of these technologies such as fixed time artificial insemination (FTAI), when correctly implemented, generally allow greater reproductive performance than natural breeding. Besides the use of frozen-thawed semen during artificial insemination, cattle recipients can also be synchronized to receive embryos (produced in vivo or in vitro) at set dates with fertility results that usually outperforms natural breeding as well as artificial insemination (AI), particularly during warm seasons and in repeat breeders cows. Altogether, the use of hormonal programs to synchronize ovulation time simplify field routine, can easily fix physiological limitations related to postpartum anestrus (beef cows), poor estrus detection efficiency due to less evident estrus signs (dairy cows), making AI and ET viable to commercial herds both in terms of results and economical returns.

The treatment of cystic ovarian disease (COD) in dairy cows is still controversial, and some researchers recommend using gonadotropin-releasing hormone (GnRH) regardless of the type of cysts. The aim of this study was to comparatively evaluate the reproductive performance of cows diagnosed with follicular or luteal cystic structures, after treatment with either buserelin acetate (GnRH agonist) or dinoprost (prostaglandin F2-alpha or PGF2α). The diagnosis was established by ultrasonographic examinations performed twice a month starting 40–45 days after calving, until the cows were diagnosed pregnant after artificial insemination. Both types of cysts were treated either with 21 µg buserelin acetate or 25 mg dinoprost, resulting in four subject groups. After treatment, the estrus rate in cows with follicular cysts treated with dinoprost (55.1%) was significantly lower (P

This paper analyses, in general, the evolution that has had the art. 7.3 LTRHA since its introduction by the Act 3/2007, 15 march. In that way, it shows which has been the legislator´s solution about the use of assisted reproduction tecniques by homosexual women couples. Likewise, it studies which resolutions on the matter have been more relevant in the last years. All this, to set out the legislator´s failure and the need to reformulate the filiation regime in face of the challenge that assisted reproduction implies.

In the field of reproductive medicine, assessment of sperm motility is a key factor for achieving successful artificial insemination, in vitro fertilization, or intracellular sperm injection. In this study, the motility of boar sperms was estimated using real-time imaging via confocal microscopy. To confirm this confocal imaging method, flagellar beats and whiplash-like movement angles were compared between fresh and low-temperature-preserved (17℃ for 24 h) porcine sperms. Low-temperature preservation reduced the number of flagellar beats from 11.0±2.3 beats/s (fresh sperm) to 5.7±1.8 beats/s and increased the flagellar bending angle from 19.8°±13.8° (fresh) to 30.6°±15.6°. These data suggest that sperm activity can be assessed using confocal microscopy. The observed motility patterns could be used to develop a sperm evaluation index and automated confocal microscopic sperm motility analysis techniques.

Feeding rumen bypass polyunsaturated fatty acids (PUFA) affects to Japanese Black cows affects their reproduction, though its influence on superovulatory response in donor cows and conception in recipient cattle has not been well studied. Here, we investigated the effects of feeding PUFA to Japanese Black cows on blood biochemistry, the numbers of ova and embryos or transferable embryos and pregnancy rate following embryo transfer (ET) to recipient Holstein heifers. PUFA (40% linoleic acid) was fed at 300 g/day in the experimental group from the last day of estrus until the day of artificial insemination for superovulatory treatment. Blood was collected on the first day of follicle-stimulating hormone administration. Total cholesterol level was significantly higher in the 15- to 19-day feeding group (117.4 mg/dl) than in the control group (95.0 mg/dl). The numbers of ova and embryos or transferable embryos were significantly higher in the 15- to 19-day feeding group than in the control group. The numbers of transferable embryos in the 15- to 19-day feeding group were significantly higher than in the 10- to 14-day feeding group. The pregnancy rate at day 60 was significantly higher in the experimental group (66.7 and 57.1%) than in the control group (51.1 and 44.0%) after transfer of fresh and frozen-thawed embryos, respectively. In conclusion, the numbers of ova and embryos or transferable embryos after superovulatory treatment increased, and the pregnancy rate after ET was higher in Japanese Black cows fed PUFA than in the control group.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonly used primary and freezing extenders (skim milk extender "INRA-82", TRIS-egg yolk extender "TEY") used in a two-step dilution approach. Furthermore the different cooling rates needed in different temperature regimes during the freezing process are being described. Cryopreservation procedures are described in case of using both specialized automated equipment and simple equipment.

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Due to the high inflow of foreign patients seeking cross-border reproductive care in Belgium and the increased number of lesbian couples and single women who call for artificial insemination with donor sperm (AID), Belgian sperm banks nowadays face a shortage in donor sperm. However, since there is no central registration system for sperm donors in Belgium, no figures are currently available supporting this statement. Therefore a study was performed to obtain a detailed overview of the sperm banking facilities in Belgium. Questionnaires were sent to all Belgian centres for assisted reproduction with laboratory facilities (n = 18) to report on their sperm banking methods. The results showed that 82% of the centres rely partially or completely on foreign donor sperm. Moreover, four of the thirteen centres that have their own sperm bank use imported donor sperm in > 95% AID cycles. Our results show that in 63% of the Belgian AID cycles imported Danish donor sperm is being used. Donor recruitment is mainly performed through the centre’s website (61%) or by distributing flyers in the centre (46%) and 9 to 180 potential donors have been recruited per centre in 2013. Eventually, 15 to 50% of these candidate donors were accepted. Different criteria for donor acceptance are handled by the centres: donor age limits range from 18-25 to 36-46 years old, and thresholds for sperm normality differ considerably. We can conclude that a wide variation in methods associated with sperm banking is observed in Belgian centres.