Semen analysis and successful artificial insemination in the St. Vincent amazon (Amazona guildingii).

The St. Vincent amazon (Amazona guildingii) is an endemic parrot on the Carribean island St Vincent. Due to poaching, trade, natural events such as hurricanes and habitat loss the species declined severely throughout the 20th century to a total number of 487 adult individuals and is currently regarded as vulnerable by IUCN. Captive breeding is attempted in terms of species conservation, but reproduction rates have been low due to reproductive problems such as mate aggression, asynchronous reproductive activity and infertile eggs. The aims of the present study were; firstly, to evaluate whether semen analysis might help to assess the fertility of males and to detect potential reasons for infertile eggs; secondly, to increase the number of offspring using artificial insemination, and as a future effect, to increase the presence of genetically valuable males in the ex-situ breeding population. For semen collection electric stimulation was used in 15 mature and healthy St. Vincent amazons with a success rate of 89% (202/227 attempts) in 14/15 males. Quality assessment of the semen included the evaluation of volume, pH, color, consistency and contaminations of the ejaculate, as well as estimation of motility, viability, morphology, concentration and total count of spermatozoa. Semen pH ranged from 6.7 to 7.5. Median sperm motility was 50% and median progressively forward motility 40%. Mean sperm concentration (x¯ ± SD) was 21,313.5 ± 22,408.8 spermatozoa/µl and mean sperm viability 66 ± 16%. Semen samples contained on average 20.5% morphologically normal spermatozoa and sperm malformations were detected mainly in the head (x¯ = 47.6%) and the tail regions (x¯ = 27.7%). Interestingly round bodies were detected in the ejaculates with a mean ratio of 0.6 round bodies per sperm. Semen analysis proved to be very useful to identify males with poor semen quality. Artificial insemination was performed 46 times in 9 females with either individual or pooled semen samples and 13 eggs from 7 females were laid afterwards. In 3 eggs, embryonic development was detected and 1 chick hatched successfully. Paternity testing confirmed the fatherhood of a one-winged semen donor male, a bird which was not able to copulate naturally. The results are very promising and underline that assisted reproduction techniques are a suitable tool for species conservation in captive breeding programs for psittacines.

Investigations on a cryopreservation protocol for long-term storage of psittacine spermatozoa using cockatiel semen as an example.

The aim of the present study was the establishment of an effective protocol for cryopreservation of psittacine semen. Therefore, pooled semen samples of 30 cockatiels (Nymphicus hollandicus) were diluted with modified Lake diluent (1:4), partitioned into four equal parts. Three portions were mixed with three cryoprotectants (dimethylacetamide, dimethyl sulfoxide, glycerol) in 4%, 8% and 12% final concentration, respectively, whereas the 4rth part served as control. Altogether, 96 incremental diluted semen samples were obtained for investigation. Each cryoprotective agent (CPA) in each final concentration was evaluated regarding sperm motility immediately after dilution and another four times every 30 min. Sperm viability was evaluated 0 and 120 min after dilution using the fluorescence stain SYBR® Green/propidium iodide. Sperm morphology was evaluated 0 and 120 min after dilution using eosin B stains. Glycerol demonstrated a lethal effect on cockatiel spermatozoa in all concentrations, whereas dimethylacetamide (DMA) in 8% final concentration proved to have the least adverse effect on semen parameters. Comparison of quick and slow freezing methods using DMA 8% revealed significantly higher rates of viable and motile spermatozoa after computer-controlled rate freezing. Two insemination experiments resulted in an egg fertility rate of 92.59% and 67.65% after artificial insemination with freshly collected semen samples, compared to 30.77% and 18.00% egg fertility rates using frozen/thawed semen. Altogether, 12 chicks hatched out of eggs inseminated with cryopreserved semen. To our knowledge, this is the first time for cockatiels to be successfully reproduced after artificial insemination using cryopreserved semen.

Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation.

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 µL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13-97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < -0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = -0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Seasonal and genera-specific variations in semen availability and semen characteristics in large parrots.

In large parrots electro-stimulation is suitable for collecting semen, and therefore, to facilitate semen examination and artificial insemination. Previous studies have detected differences in the semen collection success rate and semen parameters between psittacine genera. It remained unclear whether these differences were genera-related, seasonal variations or depend on the males' relationship status. To answer these questions, semen collection and spermatological analysis were performed for four psittacine groups (macaws, amazons, eclectus parrots and cockatoos) over 13 months. In one breeding facility, semen collection was attempted in 82 males using electro-stimulation twice monthly. A complete spermatological evaluation was performed on 435 semen samples. Volume, color, consistency, contamination and pH of semen, as well as motility, progressive motility, sperm concentration, total sperm count, viability, and morphology of spermatozoa were evaluated. Seasonality affected the collection success rate in macaws and amazons. Thereby, in amazons a distinct peak was observed several days before and around oviposition, whereas eclectus parrots and cockatoos produced semen all year round. The average sperm concentration was highest in eclectus parrots (2.7 × 106 sperm/µl) and lowest in macaws (35.6 × 103 sperm/µl). The differences in the semen collection success rate and semen parameters seem to coincide with the bird's breeding biology. The collected data allows a prognostic estimation when semen collection seems favorable, and may be taken as orientation values for semen analysis in these species.

MHC-correlated preferences in diestrous female horses (Equus caballus).

Genes of the major histocompatibility complex (MHC) have been shown to influence communication in many vertebrates, possibly with context-specific MHC-correlated reactions. Here we test for MHC-linked female preferences in the polygynous horse (Equus caballus) by repeatedly exposing 19 mares to a group of seven sexually experienced stallions. Each mare was tested four times during two consecutive reproductive cycles, twice during estrus and twice during diestrus. Male plasma testosterone concentrations were determined from weekly blood samples, and equine leukocyte antigen (ELA) class I and II alleles were determined serologically at the end of the experiments. Perception of male attractiveness was strongly dependent on estrous cycle: mean preference scores did not correlate for mares in diestrus and estrus and varied more during estrus than during diestrus. We found elevated female interests for MHC-dissimilar stallions, but only during diestrus, not during estrus. Female preferences were not significantly predicted by mean male testosterone plasma concentrations. However, testosterone concentrations changed during the 11 weeks of the experiment. By the end of the experiment, average testosterone concentration was significantly correlated to the average number of MHC alleles the stallions shared with the mares. We conclude that the MHC affects female preferences for stallions, but non-MHC linked male characteristics can overshadow effects of the MHC during estrus.

Noninvasive color Doppler sonography of uterine blood flow throughout pregnancy in sheep and goats.

In contrast to cattle or horses, uterine blood flow in small ruminants has been investigated predominantly after surgical intervention and chronic instrumentation. The objective of the present study was to investigate the clinical applicability of noninvasive color Doppler sonography to characterize blood flow in the maternal uterine artery of sheep, n = 11 (18 pregnancies) and goats, n = 11 (20 pregnancies). The following parameters were measured transrectally or transabdominally: blood flow volume, time-averaged maximum velocity (TAMV), resistance index (RI), pulsatility index (PI), Time-averaged mean velocity, impedance of blood flow (AB or systolic/diastolic [S/D] velocity ratio), peak velocity of blood flow and blood flow acceleration. Examinations started 2 weeks after breeding and continued at 2-week intervals until parturition. Outcomes for sheep and goats were similar and will be discussed together. Based on noninvasive color Doppler sonography, blood flow volume increased (approximately 60-fold, P < 0.0001) until the end of pregnancy, with a rapid increase early in gestation, and a slow increase after week 18. Time-averaged maximum velocity in the uterine artery increased (approximately 4-fold; P < 0.0001) throughout pregnancy in sheep and goats. Furthermore, for uterine artery blood flow, there was an effect of stage of pregnancy on PI and RI (P < 0.001 and P < 0.0001, respectively), both indices decreased until the end of gestation. Time-averaged mean velocity decreased from week 18 to 20 in both species. The blood flow acceleration increased (P < 0.0001) until week 16 and week 14 in sheep and goats, respectively, and then decreased until parturition. Similar to PI and RI, vascular impedance of the uterine decreased (P < 0.0001) throughout pregnancy. This is apparently the first study using noninvasive color Doppler sonography of uterine blood flow throughout physiological pregnancy in small ruminants. Clearly, this technology facilitates repeated, noninvasive assessments, with great potential for future studies.

Influence of selected (pre-)maturational parameters on in vitro development and sex distribution of bovine embryos.

The objectives of this research were to study the influence of a reduced oxygen concentration during in vitro maturation (IVM) and examine the effect of follicular glucose concentration on bovine in vitro development and sex distribution. In the first experiment, abattoir-derived cumulus-oocyte complexes (COC) were matured under 5% O2 or 20% O2. Secondly, COC were isolated and the glucose (G) concentration of each follicle was determined. COC were pooled in groups (G (< 1.1 mMol) or G (≥ 1.1 mMol)) according to the glucose content before being subjected to in vitro production (IVP). Cleavage and development rates were assessed on days 3, 7 and 8 post insemination. Blastocysts of each group were sexed by polymerase chain reaction (PCR). Expanded blastocysts were stained to assess total cell numbers and live-dead cell ratio. Cleavage and development rates stayed similar after reducing the O2 concentration during IVM. The sex ratio of embryos generated from oocytes matured under 5% O2 was shifted in favour of the female (♀: 61.9%), whereas the sex ratio of embryos belonging to the IVM 20% O2 group did not differ significantly from the expected 50:50 ratio. Neither a 'higher' nor a 'lower' intrafollicular glucose concentration influenced cleavage and development rates, cell numbers or live-dead cell ratio. Eighty five per cent (G (<1.1)) and 63.6% (G (≥ 1.1)) of the analysed embryos were female. In summary, neither a reduced O2 concentration during IVM nor selection based on follicular glucose concentrations affected the morphological quality of embryos. Although the sex distribution was shifted in favour of female embryos in all three experimental groups, more male embryos could be seen in the G (≥ 1.1) group compared with the G(<1.1) group.

Influence of social dominance on the secondary sex ratio and factors affecting hierarchy in Holstein dairy cows.

In several studies of different species, the proportions of male and female offspring did not match the statistical assumption of 50:50. Aside from environmental and genetic factors, biological parameters were found to influence the sex ratio. Three hypotheses (Trivers-Willard, advantaged daughter, and the local resource competition hypothesis) have been proposed, stating that the sex of progeny might be influenced by sex-biased maternal investment in the raising of offspring. The results of existing studies are still conflicting, even within studies on the same species. The aim of the present study was to investigate whether the dominance status of cows influences the secondary sex ratio. A herd of lactating cows was observed directly for 126 h, and a dominance index (DI) was established for 71 individuals (aged between 2.6 and 9.8 yr) for which at least 8 encounters had been observed to categorize individuals as subordinate, intermediate, or dominant. Furthermore, the number and sex of calves were assessed, and further individual characteristics of the animals (e.g., age, milk yield, body condition, and lameness scores) were analyzed for interrelationships with dominance status as well as the influence of the DI on the secondary sex ratio. The results indicated that the DI was independent of age, but a negative correlation was found between DI and lameness, whereas a positive correlation was shown for DI and body condition score. In general, more dominant animals had lower lameness scores and better body conditions. No sex ratio bias was found for primi- or for pluriparous animals and no seasonal influences were detected. The proportion of male offspring was significantly correlated with the DI of lactating cows, where animals with a higher social rank produced a lower proportion of male progeny. The pairwise comparison between dominant and subordinate lactating cows revealed a significant difference in the sex ratios of progeny, with subordinate animals having more male calves. These results indicate that the maternal dominance rank was associated with the secondary sex ratio in cattle. These observations are generally in agreement with the advantaged daughter hypothesis.

A diploid-triploid (60,XX/90,XXY) intersex in a Holstein heifer.

The diploid/triploid (60,XX/90,XXY) condition in Bos taurus is very rare and only three cases have been published previously. The present animal exhibited an aplastic vulva, penis and clitoris agenesis, a male-like urethra located in a pseudoprepuce opening between the mammary complexes and a well developed M. rectipeninus. A normal (60,XX) female karyotype was detected in lymphocyte cultures whereas uterus and tendon cells revealed a 60,XX/90,XXY mixoploidy. Quantification of X and Y chromosome-specific sequences using RT-PCR revealed extraordinary high Y chromosome equivalents in the sample recovered from the male-like transformed vestibulum vaginae suggesting a causative relationship. The pathogenesis of the missing clitoris and penis, which is contrasted by the concomitant presence of a well developed M. rectipeninus, remains difficult to explain. A chimeric origin is suggested despite the fact that microsatellite analysis of the animal's blood cells displayed no un- usual allele accumulation.

A mola hydatidosa coexistent with a foetus in a bovine freemartin pregnancy.

Molar transformations of the bovine placenta are extremly rare phenomenona and the aetiology of this genuine placental disease is still unknown. In the present study, an uncommon case of a German Holstein Friesian foetus co-twinned with a hydatidiform mole is described. Cytogenetic and fluorescence in situ hybridization analysis of cell cultures as well as prove of the presence of the SRY gene sequence revealed a heterosexual twin pregnancy. A chimeric condition of the mole was also established. In addition, an XO cell population was detected in the co-twin as well as in the mole. Upon examination of microsatellites of the parents, the mole and the co-twin an androgenetic origin of the mole is suggested, supporting the hypothesis that molar transformation of the bovine placenta may be caused by an androgenetic origin. Furthermore, the present observation demonstrates that the freemartin condition in cattle can be induced even in cases where severe placental transformations had subsequently occurred and no foetus proper could be detected at delivery.

Histone H1 and MAP kinase activities in bovine oocytes following protein synthesis inhibition.

In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 microg/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4-5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1-3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4-7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2-5 h earlier in comparison with untreated oocytes.

Ultrasonic study of follicular dynamics following superovulation in German Merino ewes.

Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be convenient and less expensive to use a single dose of FSH, but results of various trials have been controversial. We wished to investigate ovarian dynamics using ultrasonography after superovulation with a single dose of pFSH and hMG as compared with a single dose of eCG. Estrus was synchronized during the breeding season with fluorogestone acetate-containing intravaginal sponges in adult German Merino ewes (n = 38). They were superovulated with single doses of pFSH (17 mg; n = 10), hMG (600 IU FSH and 600 IU LH; n = 9) or eCG (1250 IU; n = 10) given at the time of sponge removal, or pFSH (17 mg; n = 9) given 36h before sponge removal. Follicular and luteal development were observed by ultrasonic scanning every 8 h from the gonadotrophin injection until the end of estrus, and then once daily until Day 6 after estrus. Jugular venous blood was collected starting immediately before and 1 h after superovulation treatment, then twice daily until the end of estrus and once daily for the following 7 days. Concentrations of estradiol-17beta (E2) and progesterone (P4) were measured in plasma. Differences in the follicular dynamics of the 4 superovulation groups were obvious. The functional duration of the pFSH action was estimated to last approximately 48 h, whereas eCG and hMG were active for up to 72 h. The diameter of the ovulatory follicles proved to be smaller than it was described for unstimulated ewes. Single applications of pFSH or hMG can induce a superovulatory response, although the post-estrus progesterone profile revealed a high premature luteal regression rate in the different superovulation groups. Premature corpus luteum regression could not be seen by ultrasonography at this early stage of the luteal phase, indicating that the technique may fail to detect these corpora lutea in an embryo transfer program. However, ultrasonography represents a suitable method to observe follicular dynamics following different superovulation regimens in sheep.

Estradiol-17beta and progesterone in the peripheral blood plasma of goats following superovulation with a single dose of pFSH, hMG or eCG.

During the breeding season, 42 adult German Improved Fawn nanny goats were superovulated with a single dose of pFSH, hMG or eCG at the end or a single application of pFSH 36h before the end of synchronization treatment using flugestone acetate (FGA). Plasma sampling was performed immediately before and 1h after gonadotrophin treatment, twice daily during pre-estrus and estrus and once daily during post-estrus in order to determine peripheral estradiol-17beta and progesterone levels. During that period, ovarian dynamics was followed by serial ultrasound scans (8h interval during pre-estrus and estrus and once daily during post-estrus). Estradiol-17beta profiles differed between the treatment groups exhibiting significantly positive correlations between the mean estradiol-17beta concentrations and the numbers of developing large and medium-sized follicles during estrus. The early bolus application of FSH 36h before the end of synchronization treatment induced an additional advanced estradiol-17beta peak during gestagen dominance. A sharp decrease of estradiol-17beta at the end of estrus seems to play a major role for normal luteal development. Progesterone profiles during the early luteal phase revealed high premature luteal regression. An early progesterone increase was accompanied by low premature regression rates.Although simple B-mode ultrasonography is suitable to follow follicular growth patterns and to determine the ovulation rate following different superovulation regimen endocrinological supervision is required in order to detect a premature corpus luteum insufficiency.

Ultrasonic survey of follicular development following superovulation with a single application of pFSH, eCG or hMG in goats.

During the breeding season, 42 adult German Improved Fawn nanny goats were superovulated with a single administration of pFSH, hMG or eCG at the end or a single dose of pFSH 36h before the end of estrus synchronization. Development of follicles and corpora lutea were observed by ultrasonic scanning of the ovaries every 8h from gonadotrophin treatment, until the end of estrus and once daily for the following 6 days. Differences in follicular dynamics could be realized in the four superovulation groups, and the duration of the stimulatory action following the single gonadotrophin challenge was estimated to last for 40-72h in the case of pFSH and to exceed 72h in the case of hMG and eCG treatment groups. Corpora lutea could first be detected 3 days after estrus, whereas an exact count was not possible until day 6. The ovulation rates were satisfactory, suggesting that a single injection of pFSH or hMG provides an adequate stimulus to induce a superovulatory reaction. Premature regression of corpora lutea could not be identified ultrasonographically at these early stages of the luteal phase. However, ultrasonography is a suitable method to follow follicular dynamics after superovulation and to estimate the superovulatory response in goats.

Effects of alpha-amanitin on nuclear maturation of porcine oocytes in vitro.

The effects of alpha-amanitin on cumulus enclosed and denuded porcine oocytes exposed to this heterogeneous nuclear RNA (hnRNA) synthesis inhibitor at different times (0, 0.5, 1, 2, 4, 6, 8, 12 and 20 h) after the start of culture was investigated. A further objective was to determine the sequence of nuclear progression after removing the inhibitor. The addition of 10 micrograms alpha-amanitin ml-1 to a gonadotrophin containing medium (10 iu PMSG ml-1 in TCM 199) at 0, 0.5 or 1 h after the onset of culture prevented cumulus expansion, and only 4.4, 6.4 and 3.5% of oocytes underwent germinal vesicle breakdown. This inhibitory effect was considerably reduced by delaying the addition of the drug to the culture medium for 2-8 h (2 h: 34.9%, 4 h: 53.5%, 6 h: 46.9%, 8 h: 59.2% germinal vesicle breakdown), and no inhibition of nuclear maturation was observed when alpha-amanitin was added after 12 or 20 h following explantation of the oocytes. When cumulus-oocyte complexes were cultured for > or = 2 h in inhibitor-free medium and then transferred to medium supplemented with alpha-amanitin, full cumulus expansion was observed in all cases, at the end of the 44 h culture. Denudation of the oocytes before culture in either medium supplemented with alpha-amanitin or microinjection of alpha-amanitin into the ooplasm at concentrations of 1.0 and 10.0 mg ml-1 remained without any effect on nuclear progression.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fertilization results in superovulated ewes and goats after transmural-intrauterine insemination controlled by laparoscopy]. / Befruchtungsergebnisse bei superovulierten Schafen und Ziegen nach laparoskopisch kontrollierter transmural-intrauteriner Insemination.

A comparative experiment in oestrous synchronized and superovulated ewes and does is described on which fertilization was studied following natural mating or transmural intrauterine insemination by laparoscopy. Thirty-two ewes were inseminated into each uterine horn with 50-100 microliter of fresh undiluted semen, whereas 26 ewes were mated by fertile rams. Fertilization rates of eggs recovered 5-7 days later were 93.7% (163/174) and 60.5% (78/129) respectively. Eight does were inseminated by laparoscopy while 12 does were mated by fertile bucks. Numbers of eggs fertilized of those recovered 6-7 days later were 42/47 (89.4%) in the intrauterine inseminated group and 51/60 (85%) in the naturally mated group. These results indicate that high levels of fertilization can be obtained in ewes following intrauterine insemination with fresh semen with the aid of a laparoscope and that it is possible to overcome the reduced fertilization rates after natural mating. In contrast to the ewe the intrauterine semen deposition in does did not improve the high fertilization rates obtained after natural mating.

[Current status of biotechnology in animal breeding]. / Gegenwärtiger Stand der Biotechnologie in der Tierzucht.

On the basis of the embryo transfer new and developing biotechnologies including cryopreservation of embryos, sex predetermination and sex diagnosis (sexing), in vitro fertilization, cloning and production of chimaeras and of transgenic animals are discussed.