Mitochondrial D-loop analysis reveals low diversity in Mangalica pigs and their relationship to historical specimens.

The genetic relationship between 195 Mangalica and 79 non-Mangalica pigs was studied using mitochondrial D-loop SNP genotyping. Altogether, 35 polymorphic sites and 27 haplotypes were identified. Of the haplotypes, eight and 16 are Mangalica and non-Mangalica specific, respectively, while three contain both Mangalica and non-Mangalica individuals. Genetic distance values and phylogenetic analysis indicate that Mangalica individuals are very closely related, and five haplotypes represent approximately 92% of the Mangalica pigs involved in the study, thus determining the major maternal lineages. In contrast to previous microsatellite studies, individuals of Mangalica could not be distinguished as three separate breeds using mtDNA genotyping. Comparing modern and archaeological mtDNA sequences revealed that present day Mangalica is related to pigs that lived in the Carpathian basin where postulated ancestors of Mangalica also lived. This is the first DNA-based genetic evidence to support the described breeding history of Mangalica.

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage.

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

Studies on fixed-time ovulation induction in the pig.

A technology that allows for manipulating of oestrus and ovulation, and would then also allow for fixed-time insemination, can be of great benefit for swine farms that operate using sow batch management due, at least in part, to savings in labour and the production of large batches of evenly developed pigs. Thanks to the current knowledge on endocrine regulation of follicle development and ovulation, and the availability of numerous reproductively active substances such a technology is now available. It covers procedures for synchronising oestrus based on the use of altrenogest in gilts and of batch-wise weaning in sows, for stimulating follicle development using eCG and for inducing of ovulation using hCG or LH as well as GnRH analogues. While the procedures for oestrus synchronisation stand alone, other procedures require additional treatments. If fixed-time insemination is the goal, oestrus needs to be synchronised and follicular development and ovulation induced by the use of GnRH analogues and hCG with ovulation occurring within 36-42 hrs. It is a general recommendation to inseminate those animals twice, i.e. 24 and 40 hrs after ovulation induction. However, the aforementioned technology requires healthy animals and a solid management and cannot be used to compensate for poor management.

Fertilization and early embryonic development in the porcine fallopian tube.

Fertilization and early embryo development relies on a complex interplay between the Fallopian tube and the gametes before and after fertilization. Thereby the oviduct, as a dynamic reproductive organ, enables reception, transport and maturation of male and female gametes, their fusion, and supports early embryo development. This paper reviews current knowledge regarding physiological processes behind the transport of boar spermatozoa, their storage in and release from the functional sperm reservoir (SR), and of the interactions that newly ovulated oocytes play within the tube during their transport to the site of fertilization. Experimental evidence of an ovarian control on sperm release from the SR is highlighted. Furthermore, the impact of oviductal secretion on sperm capacitation, oocyte maturation, fertilization and early embryo development is stressed.

Superovulatory ovarian response in Mangalica gilts is not influenced by feeding level.

The aim of the study was to compare how different feeding levels affect the ovarian potential of follicular development and oocyte maturation in response to superovulatory treatment in native Mangalica (M, n = 17) compared with Landrace (L, n = 20) pigs. Gilts of both breeds were fed high-energy (HI-2.5 kg) or low-energy (LO - 1.25 kg) feed during oestrus synchronization (15 days of Regumate feeding) till the time of oocyte aspiration (Day 6 after Regumate). Follicular growth was stimulated by the administration of 1000 IU equiue choriou gonadotropiu (eCG) 24 h after Regumate treatment, and ovulation was induced by injection of 750 IU human choriou gonadotropiu (hCG) 80 h after eCG administration. Ultrasound (US) investigation was done three times (4-10 h before, and 40-44 and 72-74 h after eCG administration) for the observation of follicular development. Oocyte and follicular fluid (FF) were collected endoscopically 34 h after hCG injection. Cumulus-oocyte complexes were evaluated, their morphology determined, and thereafter fixed and stained for chromatin evaluation. Oocytes were classified as meiosis-resumed (germinal vesicle breakdown, diakinesis, metaphase I to anaphase I) or matured (telophase I and metaphase II). FF concentrations of oestradiol and progesterone were measured by validated radioimmunoassays. In L gilts, differences were observed between HI and LO in the number of preovulatory follicles (32.3 +/- 10.5 vs 17.1 +/- 12.3, p < 0.05), but not in M (25.3 +/- 2.9 vs 28.8 +/- 7.3, p > 0.05). Initial follicular growth was not affected by feeding levels; however, preovulatory follicle size was larger in M (7.1 +/- 0.9 and 6.9 +/- 1.1 mm vs 5.7 +/- 0.7 and 5.5 +/- 0.8 mm; p < 0.05). No differences were obtained with relation to mature chromatin configuration in both breeds (L gilts: HI - 70% and LO-67% vs M gilts: HI - 67% and LO - 63%). A twofold higher oestradiol concentration was detected in FF of HI-M and LO-M (29.6 +/- 6.8 and 30.9 +/- 10.3 ng/ml respectively) compared with that of L (16.9 +/- 9.7 and 17.9 +/- 3.6 ng/ml, respectively; p < 0.05). The mean FF progesterone level was nearly fivefold higher in M (2020.4 +/- 1056 and 1512.2 +/- 1121.8 ng/ml) compared with L (386.2 +/- 113.7 and 298.8 +/- 125.9 ng/ml, p < 0.05). The results indicate an influence of the feeding of altered energy on the number of recruitable preovulatory follicles in modern Landrace but not in native Mangalica breed. Moreover, the follicular steroid hormone milieu differs between Landrace and Mangalica gilts but not depending on feeding levels. Oocyte maturation was not affected by diet.

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs.

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.

In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection.

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.

Comparative evaluation of the effect of antioxidants in the conservation of ram semen.

The aim of the present study was to develop a treatment supporting the membrane of ram spermatozoa. Semen of different ejaculates collected from breeding rams was mixed and samples of 10(9) sperm cells per ml and Tris-egg yolk extender were completed with the following antioxidants: alpha-tocopherol acetate (E), glutathione peroxidase (GP), Aromex (AR), resveratrol (R), resveratrol + vitamin E (RE), resveratrol + Aromex (RAR), resveratrol + GP (RGP). Peroxidation was evaluated by the analysis of malondialdehyde (MDA) during incubation for 30, 60 and 120 min at 37 degrees C as well as during a 24-h incubation at 5 degrees C. The success of preservation was checked in a 9-day-long period by observing the acrosomal defects and the motility of spermatozoa. Concentration of MDA was 4.06 nmol/10(9) spermatozoa in samples treated with 15 micrograms R while the control sample contained 69.79 nmol MDA per 10(9) spermatozoa after 24-h incubation. Following 30-, 60- and 120-min storage the concentration of MDA in control and R-treated samples was 25.89, 36.91, 49.57 and 3.69, 3.74, 3.74 nmol/10(9) spermatozoa, respectively. Moreover, a significantly higher proportion of motile sperm cells was observed in the treated than in the control samples. The frequency of acrosomal defects was lower in the treated groups than in the control. These results indicate that RAR treatment can improve the effects of ram semen preservation.

Follicular and oocyte development in gilts of different age.

The aim of the present study was to estimate follicular and oocyte development of the same gilts in three phases of their reproductive life--prepuberal gilt (6 months old), cycling gilt (9.5 months old) and primiparous sow. Follicular development was induced by injections of 1000 IU PMSG followed by 500 IU hCG 72 h later. Cumulus-oocyte-complexes (COCs) were recovered from preovulatory follicles of the left ovary, and follicular fluid (FF) from the right ovary always 34 h after hCG by endoscopy. Altogether, 19 gilts were used in the prepuberal (P) and cycling (C) trials and 12 of them in the primiparous trial (S). Altogether 168, 190 and 82 follicles were aspirated from the left ovary and 106, 125 and 42 COCs recovered (recovery rate 60.5 +/- 26.9, 62.7 +/- 20.9 and 52.9 +/- 21.8%). The average number of follicles was higher in C compared to P (19.7 +/- 6.8 vs. 15.7 +/- 6.8, p = 0.06) and to S (14.2 +/- 4.0, p < 0.05), respectively. More uniform expanded COCs were aspirated from prepuberal and cycling gilts as compared to sows (89.7 and 78.4% vs. 46.3%, p < 0.05). Furthermore, the meiotic configuration in oocytes differed (p < 0.05) between these groups (55.5 and 61.7% vs. 0% Telo 1/Meta 2). Concentrations of progesterone in FF decreased (p < 0.05) from 590.0 +/- 333.6 (P) to 249.1 +/- 72.6 (C) and 161.4 +/- 75.2 ng/ml (S). FF concentrations of oestradiol-17 beta were different between gilts and sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, p < 0.05). The progesterone/oestradiol ratio was 72.1, 15.2 and 4.7. Results indicate a different follicular and oocyte development during the investigated lifetime periods. Cycling gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows is taken into consideration at breeding. Prediction of lifetime performance based on individual ovarian reaction of prepuberal gilts is unsuitable.

A sperm tail defect associated with infertility in a goat--case report.

Semen of an infertile Dutch White (Saanenthal) goat buck was examined. Light and electron microscopic examinations showed aberrations of the sperm tails resembling the so-called Dag or Dag-like defects described in several cattle breeds. Ejaculated semen showed that virtually all of the cells had strongly coiled or broken tails, or fractured midpieces. Ultrastructural investigations by transmission electron microscopy (TEM) showed uneven distribution of the mitochondria in the midpiece. Coiled tails were encapsulated by a common membrane, and dislocated axial fibres and different membranous structures were also present. The ultrastructural characteristics of the defective sperm tails, the missing parts of the axial fibre bundle and the misalignment of the mitochondria indicate that this first case reported in goat is similar to the Dag-like defect in cattle.

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed.

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.

In vitro technologies related to pig embryo transfer.

Embryo transfer in swine (ETS) has been used for commercial and breeding application only to a limited extent. However this technique is an essential prerequisite for the application of new reproductive techniques in pigs. This paper will give an overview on steps of pig embryo transfer including selection and stimulation of donor sows, recovery of embryos, embryo handling and the transfer of recovered embryos into recipients. Furthermore the current status and further application of ET related in vitro technologies in pig production are described.

Endoscopic methods in swine reproductive research: a review.

One of the major objectives of applying clinical methods is to get a more profound knowledge of the state of an organism. With the expansion of knowledge in physiology as well as the perfection of techniques the different applications have also improved. Over the past decade it has become obvious that in swine reproductive research such techniques are needed which do not influence the physiological processes and guarantee animal welfare. The minimal invasive and non-invasive methods (transabdominal endoscopy and ultrasonography) developed in human medical practice can be successfully adapted to the requirements of swine reproductive research. The present paper reports on the experience gained with abdominal endoscopy in swine so far.

Morphology of porcine cumulus-oocyte-complexes depends on the stage of preovulatory maturation.

The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5% (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8% of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4%; P < 0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7% of COCs with expanded cumulus; P < 0.01), and 34 h after hCG, 98.3% of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2%, respectively, n = 238 oocytes; P < 0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs--88.9% diplotene, expanded COCs--53.8% metaphase II (M-II), and denuded oocytes--69.2% degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG.

Ovum pick up in swine: the influence of aspiration vacuum pressure on oocyte recovery from preovulatory follicles.

Endoscopical ovum pick up (OPU) in swine is a minimal invasive and reliable technique to aspirate oocytes from preovulatory follicles for studying intrafollicular development and oocyte maturation as well as for IVM/IVF-programs. The aim of the present study was to determine the effect of different aspiration vacuum pressures on oocyte recovery and on the morphology of cumulus oocyte complexes (COCs). Oestrus of crossbred Landrace gilts (n = 33) was synchronized by feeding altrenogest and follicular growth was stimulated with 1,000 IU PMSG 24 h after the last altrenogest application. On day 4 after PMSG application preovulatory healthy follicles of > 5 mm diameter were aspirated laparoscopically. Aspiration was carried out using a two-way cannula and an electronic aspiration pump. Five different vacuum pressures were used: 10, 17, 32, 47 and 66 ml water/min, corresponding to 30, 60, 125, 250 and 375 mm Hg, respectively. Fluids from different follicles were pooled per ovary and the morphology of cumulus oocyte complexes (COCs) was determined microscopically immediately after aspiration. COCs were classified into oocytes with intact cumulus (i.e. compact or slightly expanded COC), oocytes with corona radiata and denuded oocytes. A total of 695 follicles were aspirated and 501 COCs recovered. Increasing the aspiration pressure stepwise from 10 ml water/min up to 66 ml water/min resulted in a decrease in oocyte recovery rate. A significant higher (P < 0.05) rate of oocyte recovery (77.4% v.s. 59.8%) was achieved using a vacuum pressure of 17 ml water/min compared to 66 ml water/min, respectively. There was a tendency to reduce the portion of COCs with intact cumulus from 82 to 88% to 77% if the vacuum pressure increased to more than 47 ml water/min. A higher aspiration pressure provoked an increase (P < 0.05) in the number of denuded oocytes: 0 to 3% at 10 to 32 ml water/min, respectively, compared to 10% at 47 ml water/min to 17% at 66 ml water/min. These results demonstrate that variation in aspiration pressure affects oocyte recovery rate and COC quality. Aspiration vacuum pressure of 17 to 32 ml water/min was found to be optimal in swine both for oocyte recovery and COC quality.

Successful embryo recovery from swine by a minimal invasive technique.

An endoscopic embryo flushing technique was elaborated to make possible the minimal invasive collection of ova from swine. After experience with pig laparoscopy, the method of endoscopic embryo recovery in small ruminants was adapted to the abdominal anatomy of swine. Twelve oestrus-synchronized gilts were operated 5 days after artificial insemination. In 10 cases both uterine horns were flushed by using a Rüsch-Gold balloon catheter, flushing cannula and atraumatic grasping forceps beside the 10 mm diameter optic. The whole procedure took approximately 35-45 min. The average collection rate was 40.4%. According to the results, endoscopic embryo recovery can be a useful method in swine reproductive research and in top breeding in the future.