Ultrasonography and Reproduction in Buffalo

Ultrasonography is a simple, reliable and non-invasive imaging technique without secondary effects. Application of real time ultrasonography in veterinary practice has developed to become the most efficient diagnostic toolfor managing reproduction. The objectives of current work are to offer an overview of the uses and utility ofultrasonography for the buffalo evaluation of physiological and pathological conditions and for the application of assistedreproductive technologies. Assessment of pregnancy status and fetal viability early postbreeding to identify cows that failto conceive improves reproductive efficiency by decreasing the interval between artificial insemination services andincreasing artificial insemination service rate. Ovarian and uterine pathologies, not accurately detected via rectalpalpation, can easily be visualized by ultrasound and appropriate therapies can be implemented. Determination of fetalsex in utero is useful when coupled with a management decision that justifies the expense of fetal sexing. Developmentof integrated reproductive management systems that combine ultrasound with new and existing reproductivetechnologies will further enhance the practical applications of ultrasonography. Development of extension educationprograms to train practitioners to use ultrasound for routine reproductive examinations is a critical step toward rapidimplementation of this technology into the dairy industry

Sexing the Bovine Fetus Using Fetal Fluid Cells Recovered by Transvaginal Ultrasound-Guided Uterine Puncture

This study was aimed at using high quality ultrasonography to recover fetal fluids in early gestation (50-120 days) for the purpose of fetal sex diagnosis using the polymerase chain reaction (PCR). A transvaginal puncture technique developed for ovum pick-up was modified for fetal fluid aspiration. Where possible, and preferably, fluid was recovered from the nonpregnant horn. Fetal fluid samples were shown to contain cells and genomic DNA was extracted for sex determination by a nested, allele-specific amplification of the bovine zfx and zfy gene fragments by PCR. The accuracy and applicability of this technique was verified using abattoir material from animals of known gender. Repeated transvaginal ultrasound-guided fetal fluid recovery proved traumatic to the fetus causing fetal death and/or spontaneous pregnancy loss in six animals, five out of five cows and one out of three heifers. However, two heifers have retained live fetuses for over two weeks since the procedure. Sex diagnosis by PCR was highly accurate despite obvious maternal cell contamination of some samples. The fetal gender of all the pregnancies tested agreed with fetal sex obtained using ultrasonography or after examining the genitalia of aborted fetuses

The sabah rhino breeding programme : reproductive management of the critically endangered Sumatran rhinoceros of Borneo (Dicerorhinus sumatrensis harrissoni) as conducted by the IZW-Berlin between 2005 and 2015

Dissertação de Mestrado Integrado em Medicina VeterináriaThe Sumatran rhinoceros (Dicerorhinus sumatrensis) is on the verge of extinction. Once found throughout Southeast Asia, it stands now with less than 100 individuals scattered mainly in three national parks in Sumatra. The IZW-Berlin has been collaborating with BORA/SWD through the use of advanced imaging and assisted reproduction techniques in wild-caught Bornean rhinoceroses (D. s. harrissoni) held at the BRS. Ultrasonographic examinations and reproductive procedures conducted between 2005 and 2015 in 2 male and 3 female rhinoceroses were retrospectively analysed in order to infer on reproductive condition and its evolution, outcome of procedures and impact of interventions. Furthermore, the study aimed at developing a detailed description of ultrasonographic findings and identify evidence to further elucidate on the estrous cycle of the species. A total of 17 working visits were included in the descriptive analysis with 56 reproductive assessments by ultrasonography, 8 semen collections by electroejaculation, 10 hormonal treatments, 1 artificial insemination, 5 oocyte collections, 3 intracytoplasmic sperm injections, 4 techniques for the removal of endometrial cysts and 1 hydrosalpinx aspiration. The detailed description of findings provides new technical information on numerous anatomical structures of both males and females, and constitutes the first report of various procedures in the species. Notably, several hypotheses are considered and put forward for future investigation. Results revealed that no animal suffered negative consequences from the repeated and sometimes invasive interventions. While the older female was found to have entered early reproductive senescence, the two young females were reproductively active but showed a severely impaired reproductive tract, namely affected by endometrial cystic hyperplasia and uterine leiomyomas. These findings were associated to the phenomenon of asymmetric reproductive ageing and a presumptive relationship with lack of breeding and low densities in the wild is reported. Ultrasonography further revealed interesting and novel findings on estrous cyclicity, which in combination with observed pathological changes may explain the lack of success of most reported procedures. In regard to the males, semen collections were successful but semen quality proved to be very low, and hypotheses of this being related to husbandry issues and/or pathological changes are reported.RESUMO - Programa de Reprodução do Rinoceronte de Sabah: maneio reprodutivo do rinoceronte-de-Sumatra do Bornéu (Dicerorhinus sumatrensis harrissoni) criticamente ameaçado de extinção, como conduzido pelo IZW-Berlim entre 2005 e 2015 - O rinoceronte-de-Sumatra (Dicerorhinus sumatrensis) encontra-se à beira da extinção. Outrora presente em todo o sudeste asiático, está agora reduzido a menos de 100 indivíduos separados por três parques nacionais na Sumatra. O IZW-Berlim tem colaborado com a BORA/SWD através do uso de técnicas avançadas de imagem e reprodução assistida nos rinocerontes do Bornéu (D. s. harrissoni) capturados do meio selvagem e mantidos no BRS. Exames ultrassonográficos e procedimentos reprodutivos realizados entre 2005 e 2015 em 2 machos e 3 fêmeas foram analisados retrospetivamente para inferir sobre condição reprodutiva e sua evolução, resultado dos procedimentos e impacto das intervenções. Adicionalmente, procurou-se desenvolver uma descrição detalhada dos achados ecográficos e identificar evidências que ajudem a compreender o ciclo éstrico da espécie. Um total de 17 visitas foram incluídas na análise descritiva com 56 avaliações reprodutivas por ultrassonografia, 8 recolhas de sémen por electroejaculação, 10 tratamentos hormonais, 1 inseminação artificial, 5 recolhas de oócitos, 3 injeções intracitoplásmicas de espermatozóides, 4 técnicas para a remoção de quistos endometriais e 1 aspiração de hidrossalpinge. A descrição detalhada dos achados fornece nova informação técnica sobre numerosas estruturas anatómicas de machos e fêmeas, e constitui o primeiro registo de diversos procedimentos na espécie. Notavelmente, várias hipóteses são avançadas para investigação futura. Os resultados revelaram que nenhum animal sofreu consequências negativas das intervenções repetidas e por vezes invasivas. Enquanto a fêmea mais velha se encontrava em senescência reprodutiva precoce, as duas fêmeas mais jovens exibiam atividade reprodutiva mas também um aparelho reprodutivo extremamente afetado por hiperplasia quística do endométrio e leiomiomas uterinos. Estes achados foram associados ao fenómeno do envelhecimento reprodutivo assimétrico, e uma relação presuntiva com ausência de atividade reprodutiva e baixas densidades em meio selvagem é reportada. A ultrassonografia revelou ainda novos achados em relação ao ciclo éstrico, que em conjugação com as alterações patológicas observadas poderão explicar a falta de sucesso da maioria dos procedimentos relatados. Em relação aos machos, recolhas de sémen foram realizadas com sucesso mas revelaram uma qualidade muito baixa, e hipóteses desse facto estar relacionado com problemas de maneio e/ou alterações patológicas são reportadas.N/

Semen quality detection using acoustic wave sensors

Artificial insemination (AI) is a widely used part of the modern agricultural industry, with the number of animals inseminated globally being measured in the millions per anum. Crucial to the success of AI is that the sperm sample used is of a high Quality. Two factors which determine the quality of the sample are the number of sperm present and their motility. There are numerous methods used to analyse the quality of a sperm sample, but these are generally laboratory based, expensive and in need of a skilled operator to perform the analysis. It would, therefore be useful to have a simple and inexpensive system which could be used outside the laboratory, immediately prior to the insemination of the animal. Presented in this thesis is work developing a time of flight (ToF) technique which makes use of a quartz crystal microbalance (QCM), operating at 5 MHz, as the sensing element. Data is shown developing a device where a 50 μl sample of boar sperm is added to a liquid filled swim channel, which the sperm are allowed to self-propel down and attach to the surface of a QCM at the end. The attachment of the sperm to the surface causes a measurable frequency decrease in the QCM, aproximately 50 Hz. An average effective mass measurement was made using a QCM and gave a value of 8 ± 5 pg per sperm, which was used in conjunction with the frequency change to determine the number rate of sperm reaching the QCM

Insights from Animal Reproduction

The chapters in this volume of "Insights from Animal Reproduction" address several, particular hot topics in the field of reproduction. The book begins with a comprehensive overview of the cryopreservation of sheep-produced embryos. The following chapter revises the assisted reproductive techniques available for South American wild mammals. Chapter 3 presents the technical procedures necessary to produce transgenic goats. Chapter 4 provides a comprehensive revision of the major molecular determinants of litter size in prolific species. Chapter 5 examines the germ cell determinant transmission, segregation, and function using the zebrafish as a model for germ cell specification in the embryo. Chapter 6 summarizes the current understanding of the molecular and cellular mechanisms regulating the early stages of folliculogenesis. Chapter 7 examines the sperm motility regulatory proteins as a tool to enhance sperm quality in cryopreservation processes. Chapter 8 discusses contemporary knowledge on the effects of extremely low frequency magnetic fields (ELF-MF) on male reproductive function in rodents. Chapter 9 highlights the importance of the cytogenetic evaluation in searching for causes of infertility of phenotypically normal animals, as well as individuals with an abnormal sex development. The last chapter provides evidence that other uterine diseases may be hidden behind the clinical diagnosis of pyometra that in some case may have a poor outcome

Developmental competence of equine oocytes after ICSI : Implications on technical, morphological and cellular aspects

Using equine intracytoplasmic sperm injection (ICSI) procedure, the present study was performed, (i) to study the effect of assisted oocyte activation with calcium ionophore A23187 on developmental competence of oocytes, (ii) to re-evaluate the effect of cumulus morphology on meiotic and developmental competence of oocytes, (iii) to investigate the effect of glucose-6-phosphate dehydrogenase (G6PD) activity on meiotic and developmental competence of oocytes and (iv) to analyze zona pellucida (ZP) properties of equine oocytes of different quality and maturational status, quantitatively by polarization light microscopy. One hour after ICSI, injected oocytes were categorized into two groups: those treated with calcium ionophore A23187 and non-treated group. Cleavage and blastocyst rate were significantly (PDie Entwicklungskompetenz equiner Eizellen nach ICSI: technische, morphologische und zelluläre Aspekte inbegriffen Die vorliegende Studie untersucht den Effekt der assistierten Eizellaktivierung mit Calcium Ionophor A23187 equiner Embryonen nach der intrazytoplasmatischen Spermainjektion (ICSI) auf die Entwicklungskompetenz. Es wurde eine Re-evaluierung des Einflusses der Kumulusmorphologie, der Glucose-6-Phosphat-Dehydrogenase (G6PD) Aktivität und der Beschaffenheit der Zona pellucida mit Hilfe der Polarisationslichtmikroskopie im Hinblick auf die präimplantative Entwicklungskompetenz durchgeführt. Eine Einteilung in zwei Gruppen wurde eine Stunde nach ICSI vorgenommen, wobei nur eine Gruppe mit Calcium- Ionophor A23187 behandelt wurde. Die mit Calcium-Ionophor A23187 behandelten Eizellen zeigten signifikant höhere Teilungs- und Blastozystenraten (P < 0,05) als jene, die nicht behandelt wurden. Die Kumulusmorphologie der ungereiften Eizellen (expandierter Kumulus (Ex) vs. kompaktierter Kumulus (Cp)) zeigte ebenfalls signifikante Unterschiede (P<0,05) in der Maturationsrate und der Blastozystenrate nach ICSI. Zur Messung der G6PD Aktivität der Eizellen wurden diese mit Brilliant Cresyl Blue (BCB) gefärbt. Der prozentuale Anteil der Eizellen mit einer geringeren G6PD-Aktivität und blauem Cytoplasma war in der Gruppe der Ex Eizellen signifikant höher (P<0,01) als in der Gruppe der Cp Eizellen. Des Weiteren wurden signifikant höhere (P<0,05) Maturations- und Entwicklungsraten der BCB+ Eizellen beobachtet verglichen mit Eizellen mit hoher G6PD Aktivität. Mit Hilfe der Polarisationslichtmikroskopie wurde die Zona pellucida der Eizellen mit unterschiedlicher Entwicklungskompetenz (Ex vs. Cp und BCB+ vs. BCB-) und in verschiedenen Reifungsstadien (Immature Eizellen und mature Eizellen mit und ohne Polkörper) vermessen. Unsere Ergebnisse zeigen, dass die Ex Eizellen verglichen mit den Cp Eizellen und die BCB+ Eizellen verglichen mit den BCB- Eizellen eine signifikant (P<0,05) dickere Zona pellucida und eine höhere Doppellichtbrechungsintensität aufweisen. Zusätzlich hatten die Eizellen, die nach der in vitro Maturation keinen Polkörper ausschleusten, eine signifikant (P<0,05) dickere Zona pellucida und eine signifikant (P<0,05) höhere Doppellichtbrechungsintensität als immature Eizellen und Eizellen, die einen Polkörper ausschleusten. Zusammenfassend zeigte sich dass sich die Aktivierung der Eizellen nach ICSI mit Calcium-Ionophor A23187 äußerst positiv auf die Entwicklung der equinen Embryonen auswirkt. Kumulusmorphologie wie auch G6PD Aktivität sind verlässliche Indikatoren für die Entwicklungskompetenz. Ebenfalls könnten die Dicke und die Struktur der Zona pellucida, gemessen an Hand der Doppellichtbrechungsintensität, als neue Methode zur Bestimmung des Entwicklungspotentials equiner Eizellen dienen

From sperm selection to embryo development: New insights into key assisted reproductive technologies in the horse

Since the early 1990s, sport horse breeding has increasingly adopted assisted reproductive technologies (ARTs), starting with artificial insemination (AI) and followed by embryo transfer and in vitro embryo production (IVP). AI is preferentially performed with chilled transported semen, which is limited by optimal storage times of 24-48 hours. We investigated the impact of prolonged cold storage (96 hours at 5 °C) on sperm quality and found time-related increases in plasma membrane fluidity and intra-cellular Ca2+, indicators of premature capacitation, that probably explain diminishing fertility. While IVP by intracytoplasmic sperm injection (ICSI) dramatically reduces the number of ‘fertile’ spermatozoa required, it bypasses natural sperm selection processes that prevent sperm with damaged DNA fertilizing the oocyte. We developed a 3-layer density-gradient centrifugation technique that selects a population of motile, morphologically normal spermatozoa with intact DNA better suited for ICSI. We also investigated the impact of IVP on early cell lineage segregation and found IVP blastocysts to contain fewer, more widely dispersed epiblast cells. Epiblast cell dispersal may explain why IVP embryos are less viable than in vivo embryos, but more likely to give rise to monozygotic twins. Fortunately, transferring IVP blastocysts into a mare's uterus triggered rapid epiblast compaction. Whereas IVP embryos are routinely cryopreserved, freezing larger (>300 µm diameter) in vivo-derived blastocysts has proven challenging. Recently, it transpired that puncturing large blastocysts and aspirating the blastocoel fluid improves their freezability; we further demonstrated that punctured embryos survive vitrification better than slow-freezing. Overall, this thesis highlights areas for improvement within equine ART

Assessment of laser-assisted micromanipulation procedures in a commercial bovine in vitro production laboratory

In vitro production (IVP) of bovine embryos is increasing yearly and is rapidly becoming the most commonly-used tool in cattle breeding. The drive for more efficient food production requires accelerated dissemination of superior cattle genetics. The implementation of advanced techniques such embryo biopsy and laser assisted hatching (LAH) with IVP embryos facilitates early genetic selection and could enhance pregnancy rates following embryo transfer. The nature and extent of chromosomal errors can also be established from embryo biopsies, and this could also improve pregnancy outcomes following embryo transfer. However, the techniques of bovine embryo biopsy and LAH are laborious, time consuming, utilise expensive equipment and require a high degree of technical skill. This thesis describes a series of experiments which sought to develop easy and robust methods for embryo biopsy and LAH in a commercial laboratory setting. It assessed the survivability and ‘hatchability’ of embryos compared to those that were not manipulated

Final follicular maturation in the cow and its effects on the developmental potential of the oocyte

The use of assisted reproduction techniques can generate up to 27 (superovulation, SO) or 50 (in vitro embryo production, IVP) calves per cow per year instead of only one calf per cow per year after normal mating. That is due to the possibility to use more than one oocyte per estrous cycle; namely on average 15 (SO) and 60 (IVP). However, using these techniques, the efficiency per used oocyte decreases dramatically. Instead of the efficient use of oocytes during natural mating, only 30% efficiency is reached with SO. Furthermore, IVP generates only 8 calves per 100 used immature oocytes. This loss in efficiency might be due to deviations that occur during the final maturation of the oocytes. In this thesis concepts of final follicular maturation in unstimulated normally cyclic cows are applied to maturation during SO and IVP in order to gain better understanding of deviations in follicle and oocyte development using these techniques. Also preovulatory follicular development after SO is used as a model to study maturation in vivo. Chapter 1 introduces the subject. Deviations in preovulatory follicular development as induced using SO or as a result of oocyte collection and maturation during IVP are discussed against the background of the course of follicular maturation in unstimulated normally cyclic cows. In cows treated for SO the period of preovulatory follicular development is reduced compared to this period in unstimulated normally cyclic cows. Also, after SO an asynchrony of development is found within the population of stimulated follicles as well as between the follicle and its oocyte. For IVP, oocytes are usually collected from 3-6 mm follicles. These follicles lack part of the processes of follicular growth and selection. Furthermore, during in vitro maturation (IVM) of the collected immature oocytes, deviations in cytoplasmic maturation are frequently found. In chapter 2, the effect of prolongation of the period of preovulatory follicular development after SO on the heterogeneity of the population of preovulatory follicles and their oocytes with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos was investigated. In eCG-stimulated heifers, the spontaneous occurrence of the LH surge was suppressed with a norgestomet ear implant and at a later time a LH surge was induced using GnRH. The protocol resulted in a LH surge at the desired time in 100% of the cases. Prolongation of the period of preovulatory follicular development from 42.4 to 53.8 h increased ovulation rates with 25%. It was suggested that the heterogeneity of the follicular population, as is present in normally stimulated heifers at the time of the spontaneous LH surge, was reduced. The increased ovulation rates did not coincide with an increased number of embryos at day 7 after fertilization. The treatment with norgestomet did not adversely affect Chapter 8?Chapter 8 122 final maturation and fertilization. However, the treatment might have disturbed early embryonic development by altering the secretory activity of the cells of the epithelium of the oviduct. The superovulation protocol with LH surge induction (chapter 2) was used in the studies described in chapter 3 and 4 to obtain oocytes at a fixed stage of development in vivo. In chapter 3, it was investigated to what extend IVM contributes to limiting yields of viable embryos in currently used IVP programs. The use of in vivo matured oocytes, collected from eCG-stimulated heifers at 22-24 h after the LH surge, instead of in vitro matured oocytes collected from 2-8 mm sized follicles, for IVF and IVC improved blastocyst formation and hatching with 100%. Additionally, also the progress of embryonic development was different for the two groups of oocytes; both blastocyst formation and hatching of blastocysts progressed slower for in vivo matured oocytes than for in vitro matured oocytes. The decreased embryonic development after IVM (chapter 3) might be due to the maturation conditions per se or to a difference in startcompetence of the oocytes collected from 2-8 mm sized follicles when compared to the oocytes from preovulatory follicles generated in eCG-treated cows. In chapter 4 the significance of the conditions during maturation for efficient IVP was tested using oocytes for in vivo maturation and IVM which presumably had an equivalent startcompetence. Therefore, heifers were stimulated with eCG using the same protocol as described in chapter 2. From part of the heifers, oocytes from preovulatory follicles were collected at the presumptive time of the LH surge and subsequently subjected to IVM. From the other heifers, oocytes from preovulatory follicles were collected 22-24 h after the occurrence of an induced LH surge. Both groups of oocytes were subjected to IVF and IVC. Blastocyst formation and hatching rates were significantly lower after in vitro maturation than after in vivo maturation of the oocytes. It was concluded that the conditions during IVM are an important factor responsible for limited yield after IVP. In this study, the progress of embryonic development was similar for both groups of oocytes and was conform to that as observed for the in vivo matured oocytes in chapter 3. Since in that study in vitro matured oocytes from 2-8 mm follicles developed at a faster rate than in vivo matured oocytes from preovulatory follicles it was suggested that oocytes undergo certain changes during follicular development from 2-8 mm to preovulatory stage at onset of final maturation which may be involved in programming embryonic development. Both in the studies described in chapter 3 and 4, a large variation in blastocyst formation between individual heifers was found, which is probably due to differences in response to superovulatory treatment. When in the group of heifers donating the in vitro matured oocytes (chapter 4) heifers with exceptional follicular function on the basis of estradiol-17ß and progesterone concentrations in the follicular fluid (FF) were excluded, rates of blastocyst formation increased indicating that the steroid producing capacity of the follicular wall influences oocyte quality. In chapter 5 and 6 preovulatory follicular development after eCG-treatment for SO was used as a model to study the possible role of the insulin-like growth factor (IGF)?Summary 123 system during final maturation in vivo. Earlier results from in vitro studies suggested a stimulatory role of IGF-I on growth and differentiation of granulosa cells and on final maturation of the oocyte. IGF binding proteins (IGFBPs) are suggested to decrease bioavailability of IGF-I but also individual functions of these IGFBPs on follicle and oocyte maturation can not be excluded. In chapter 5, levels of IGF-I and IGFBPs in the FF of eCG-stimulated follicles with normal and deviant follicular function on the basis of steroid hormones in the FF were compared at several times during final maturation. Final maturation in eCG-stimulated preovulatory follicles with normal function was characterized by rather constant levels of IGF-I and IGFBP3 in the FF. Low molecular weight IGFBPs (LMW IGFBPs, i.e. IGFBP2, -4 and -5) were absent in FF at the time the oocyte undergoes germinal vesicle breakdown (GVBD) and reaches metaphase I (MI). Just prior to ovulation, in 15% of the FF of the follicles with normal function, LMW IGFBPs were found. While the levels of IGF-I and IGFBP3 in the fluid of deviant follicles were not different when compared to eCG-stimulated follicles with normal function, the presence of LMW IGFBPs was different in these follicles; at the onset of final maturation, 35% of the deviant follicles contained LMW IGFBPs. All these follicles, except one, were classified deviant due to too low estradiol-17ß concentrations, probably a sign of atresia. During final maturation, LMW IGFBPs were occasionally found (GVBD, MI) or absent (just prior to ovulation) in deviant follicles. It was concluded that only a permissive role of IGF-I in final maturation can be expected. It was also concluded that, as in unstimulated, normally cyclic cows, low estradiol-17ß concentration in the FF of large follicles (> 8mm) coincided with the presence of LMW IGFBPs. The appearance of LMW IGFBPs in the FF of follicles with normal follicular function just prior to ovulation indicated a different role of the IGF system during ovulation and corpus luteum formation. Because different IGFBP patterns were found in the FFs of eCG-stimulated follicles with deviant follicular function and of concurrently developing follicles with normal follicular function, eCG stimulated follicles were used to study the regulation of the presence of IGFBPs during preovulatory follicular development (Chapter 6). Using reverse transcriptase-polymerase chain reaction (RT-PCR), mRNA levels for LMW IGFBPs were analyzed (semi-quantitatively) in eCG-stimulated preovulatory follicles at the onset of maturation with normal or deviant function, on the basis of estradiol-17ß concentrations in their FF. No or small differences in gene expression were found. Therefore, it was concluded that the earlier found difference in the presence of LMW IGFBPs in FF could not be explained by a difference in gene expression for these IGFBPs in the wall of the follicle. Thus, regulation of the presence of these proteins has to take place at the level of translation or protein degradation. Finally, in chapter 8, the results of the performed experiments are discussed against the background of the current knowledge and existing hypotheses on final follicular maturation. Possible directions of future research in order to improve the efficiency of assisted reproduction are discussed