Alternation of inverse problem approach and deep learning for lens-free microscopy image reconstruction.

A lens-free microscope is a simple imaging device performing in-line holographic measurements. In the absence of focusing optics, a reconstruction algorithm is used to retrieve the sample image by solving the inverse problem. This is usually performed by optimization algorithms relying on gradient computation. However the presence of local minima leads to unsatisfactory convergence when phase wrapping errors occur. This is particularly the case in large optical thickness samples, for example cells in suspension and cells undergoing mitosis. To date, the occurrence of phase wrapping errors in the holographic reconstruction limits the application of lens-free microscopy in live cell imaging. To overcome this issue, we propose a novel approach in which the reconstruction alternates between two approaches, an inverse problem optimization and deep learning. The computation starts with a first reconstruction guess of the cell sample image. The result is then fed into a neural network, which is trained to correct phase wrapping errors. The neural network prediction is next used as the initialization of a second and last reconstruction step, which corrects to a certain extent the neural network prediction errors. We demonstrate the applicability of this approach in solving the phase wrapping problem occurring with cells in suspension at large densities. This is a challenging sample that typically cannot be reconstructed without phase wrapping errors, when using inverse problem optimization alone.

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Cryopreservation of in vitro produced bovine embryos: effects of lipid segregation and post-thaw laser assisted hatching.

The objective was to determine if lipid segregation, with or without post-thaw laser assisted hatching (LAH) of in vitro produced (IVP) bovine embryos, would enhance in vitro survivability and development 24 h post-thaw. On Day 6 of culture (Day 0 = IVF), in vitro produced bovine embryos were divided into three developmental stages: 32-cell (n = 78), compact morula (CM n = 223), and blastocyst (n =56). Embryos within each stage were allocated to the following treatments prior to cryopreservation in 1.5M ethylene glycol: no treatment (Control), 7.5 µg/mL Cytochalasin B for 20 min (CB), or CB with centrifugation (16,000 × g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Immediately post-thaw, one-half of the CBCF and Control groups were subjected to zona pellucida drilling (LAH), using the XY Clone® system, creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured for 24 h and evaluated. No treatment differences were observed for either post-thaw survival or 24 h development. Within the CM stage, CBCFLAH and LAH exhibited a greater number of both total and live cells than Control (total: 69.4, 69.3, 53.0, live: 56.4, 54.7, 39.3 respectively; P < 0.05). In conclusion, LAH post-thaw alone or in combination with CBCF improved embryo viability following cryopreservation.

Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares.

Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in resulting blastocysts, and were compared to those for embryos derived in vivo from control or exercised mares. Exposure of oocytes to heat at the onset of in vitro maturation did not affect any measured end point. However, exposure to 42 degrees C late in maturation culture reduced rates of oocyte nuclear maturation for both the 2 h (43/105 (43%) compared to control 70/103 (68%); P < 0.01), and 4 h (47/106 (44%) compared to control 60/103 (59%); P < 0.05) groups. Additionally, late heat exposure reduced development to morulae and blastocyst stages after intracytoplasmic sperm injection (ICSI; 18/89 (20%) compared to control 43/128 (34%); P < 0.05). Seven days after oocyte heat exposure, resultant blastocysts had a higher abundance of HSPA1A gene transcripts, relative to those for 18S rRNA. In vitro-produced embryos and lower-quality in vivo-produced embryos had significantly higher relative HSPA1A mRNA (lower 18S rRNA) concentrations than did higher-quality in vivo-produced embryos. The authors concluded that equine oocytes were sensitive to heat during late in vitro maturation, and responded to thermal shock with an increased ratio of HSPA1A:18S gene expression that was measurable in the resulting blastocyst. Embryos produced in vitro (including controls) had increased levels of HSPA1A mRNA relative to 18S rRNA compared to in vivo-produced embryos, suggesting a response to environmental insult.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Embryo recovery from exercised mares.

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 degrees C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F(2alpha) was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 degrees C to a mean of 39.9 degrees C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8+/-0.5 compared with 41.5+/-0.5mm diameter; P<0.05), and had an increased time from PGF(2alpha) administration to subsequent ovulation (8.47+/-0.337 compared with 9.27+/-0.294 d; P<0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P<0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P=0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.

Role of cumulus cells on in vitro maturation of canine oocytes.

The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.

Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro.

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P<0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovaries of bitches in oestrus and dioestrus had higher rates of maturation to metaphase II than did oocytes from bitches at pro-oestrus or anoestrus (P<0.01). In Expt 3, no differences were observed in rates of maturation among treatment groups. Rates of maturation to metaphase II of oocytes from bitches in dioestrus were significantly higher than those from bitches in pro-oestrus (P<0.01). These results indicate that supplementation of culture medium with progesterone either during maturation or during meiotic arrest before maturation does not increase the rate of IVM of canine oocytes. However, stage of oestrous cycle is a key factor in the selection criteria for meiotically competent canine oocytes for use in in vitro experiments.

Effects of oocyte culture density on meiotic competence of canine oocytes.

This study was conducted to determine a suitable ratio of oocytes to medium for in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) collected from bitches at anoestrus and dioestrus and to examine the meiotic competence of COCs cultured singly or in different group sizes. In the first experiment, different numbers of COCs (5, 10, 15 and 20 per drop) were cultured for 72 h in 100 microl drops of maturation medium. The meiotic competence of oocytes from ovaries at anoestrus was affected by the number of COCs incubated, whereas at dioestrus, the incubation number of COCs had no effect. In the second experiment, COCs were cultured singly or in different group sizes for 72 h by suitable oocyte density according to the reproductive cycle of the donor. In the anoestrous group, 1, 5 and 10 COCs were cultured in 10, 50 and 100 microl drops of the medium (10 microl per COC), respectively. In the dioestrous group, 1, 5 and 15 COCs were cultured in 7, 35 and 105 microl drops of the medium (7 microl per COC), respectively. There were no differences in the proportions of oocytes reaching metaphase II among the different group sizes in each stage of the reproductive cycle of the donor. The results indicate that the influence of oocyte density on the meiotic competence of oocytes differs according to the stage of the reproductive cycle of the donor. Moreover, the group sizes have no effect on the meiotic competence of oocytes cultured at suitable oocyte density according to the reproductive cycle of the donor.

Potential for cloning dogs.

The aim of this study was to determine whether nuclear transplantation could be used to clone a dog using donor nucleus cells collected from an adult female. Fibroblasts obtained from skin biopsies were fused with enucleated bovine or canine oocytes. The resulting cloned embryos were cultured in vitro to monitor embryonic development. A proportion of the resulting embryos was transferred into surrogate bitches for development to term. When canine oocytes were used as recipient ova for canine fibroblasts, 23% of the resulting embryos cleaved at least once after culture in vitro. Five cloned embryos were transferred into three synchronized recipient bitches, but no pregnancies resulted. When bovine oocytes were used as recipinets for canine fibroblasts, 38% cleaved to the two- to four-cell stage and 43% cleaved to the eight- to 16-cell stage. Forty-seven of these embryos were transferred into four recipient females, resulting in a single conceptus that ceased development at about day 20 of gestation. The desire for cloned dogs is considerable and will undoubtedly incite the development of successful methods for cloning companion animals. However, significant investment into additional research is required, especially in the areas of in vitro maturation of oocytes and control of the oestrous cycle of bitches.

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei.

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.

A simple salt solution medium supplemented with yolk plasma and lactate (YPLM) supports development of preimplantation bovine embryos in vitro.

This study investigated the embryotropic potential(s) of egg yolk (EY) or its fractions, yolk plasma (YP) and yolk granules (YG), in culturing bovine embryos in vitro and substituting for protein (FCS, BSA, and BME-E and MEM-NE amino acids) and energy (glutamine, pyruvate and L-lactate) supplements commonly added to culture medium. In the first set of experiments (Experiment 1, 2 and 3) CR1aa with buffalo rat liver (BRL) cells were used as a co-culture system. The addition of 2.5% or 5% EY significantly increased (P<0.05) blastocyst percent over the BRL control (48.3% and 52.4% vs. 32.5%, respectively). The addition of 5% EY in the absence of FCS and BSA resulted in percent development to blastocysts and hatched blastocysts similar (P>0.05) to those of the BRL control (37.6% and 57.4% vs. 51.5%, 22.7% and 39.5%, respectively). The supplementation of the BRL control with 5% YP compared to that of EY resulted in comparable (P>0.05) percentages of blastocysts and hatched blastocysts (39.0% and 51.6% vs. 40.0% and 58.3%, respectively). In the second set of experiments, the embryotropic potential of YP was examined using a cell-free culture system and a simple salt solution (SS) of NaCl, KCl and NaHCO3 as the base medium. The supplementation of an energy-supplemented cell-free simple salt solution (E-SS) with 5% YP in the absence of supplemental protein resulted in percent development into blastocysts and hatched blastocysts comparable (P>0.05) to those of a BRL control (39.2% and 15.8% vs. 37.1% and 22.2%, respectively). The addition of YP to the simple salt solution with hemicalcium L-lactate as the only supplemented energy ingredient resulted in percentages of blastocysts and hatched blastocysts similar (P>0.05) to those obtained by the supplementation of all energy sources (27.4% and 15.6% vs. 36.4% and 14.0%, respectively). Increasing hemicalcium L-lactate level from 5 to 10, 20 or 25 mM resulted in a significant decrease (P<0.05) in percent development into blastocysts (36.5% vs. 24.8%, 11.6% and 6.7%, respectively). In conclusion, YP, with the advantage of being clearer than EY, is capable of sustaining embryo development to the blastocyst stage in a simple salt solution of NaCl, KCl and NaHCO3 supplemented with hemicalcium L-lactate.

Plasma vitamin E and other analyte levels in Nepalese camp elephants (Elephas maximus).

Plasma concentrations of alpha-tocopherol (vitamin E) and other analytes in Asian elephants (Elephas maximus) in Nepal were determined during typical work camp management of the elephants. Elephants foraged for food for 4-6 hr each day under the control of mahouts and were also provided daily with cut forage and supplements of unhusked rice, cane molasses, and salt. Blood samples were taken monthly for 1 yr without chemical restraint from 26 female elephants in four camps. Elephants were 6-60+ yr of age. Mean (+/-SEM) alpha-tocopherol concentration was 0.77+/-0.047 microg/ml, with a range of 0.23-1.57 microg/ml. Subadults had lower concentrations than did older elephants, and there were significant differences in mean concentrations from different camps and in mean monthly concentrations. Plasma alpha-tocopherol concentration appears to vary widely between individuals, and a single value of <0.3 microg/ml is not sufficient to diagnose incipient vitamin E deficiency. Mean (+/-SEM) plasma retinol (vitamin A) concentration was 0.063+/-0.003 microg/ml with a range of 0.01-0.12 microg/ml. Subadults had higher concentrations than did older elephants, and mean retinal values differed significantly among camps. Beta-carotene was not found in plasma. Twenty-five other analytes determined or derived were generally similar to those reported in other Asian and African (Loxodonta africana) elephants. Estimates of nutrient intake, based upon diet composition, suggested that dietary concentrations of zinc and sodium may have been marginal, but the absence of signs of any nutrient deficiencies indicates that dietary husbandry in these elephant camps was generally satisfactory.

Ultrasonographic evaluation of the effects of exogenous gonadotropins on follicular recruitment and ovulation induction in the Atlantic bottlenose dolphin (Tursiops truncatus).

Transabdominal ultrasonography and serum steroid concentrations were used to evaluate the effects of exogenous gonadotropin administration on ovarian activity of two anestrous bottlenose dolphins (Tursiops truncatus). The gonadotropin used for follicular recruitment was PG600, which has 400 IU equine chorionic gonadotropin (eCG) and 200 IU human chorionic gonadotropin (hCG) activity per 5 ml. Ovulation induction was attempted with hCG. PG600 was administered in two doses of 20 ml (1,600 IU eCG and 800 IU hCG) and 12.5 ml (1,000 IU eCG and 500 IU hCG), respectively, 48 hr apart on days 0 and 2. On day 6, 1,500 IU of hCG was administered. Progesterone and total immunoreactive estrogens were determined before and after the gonadotropin administration. Bilateral ovarian ultrasonographic exams were performed daily on days 4-9 and on day 22. Serum immunoreactive estrogen concentrations were greater than the pretreatment concentrations after day 4 for both dolphins and remained elevated for the rest of the study. Serum progesterone concentrations rose above 1 ng/ml 2 days after hCG treatment and remained elevated for the rest of the study. Small antral follicles (< 0.5 cm) were initially observed bilaterally in both dolphins on day 4. In both animals on day 9, there were > 12 follicles/ovary, ranging from 0.5 to 1.5 cm. By day 22, the multiple follicles ranged from 0.5 to 4.5 cm in diameter. No ultrasonographic evidence of luteal formation was observed. The results indicate that 1) transabdominal ultrasonography can be used to detect and follow follicle growth in bottlenose dolphins; 2) bottlenose dolphins are sensitive to exogenous gonadotropins (multiple follicular recruitment of follicles occurred); and 3) until further ultrasonographic studies can be conducted to evaluate the effects of titrated doses of exogenous gonadotropins, these protocols should be considered unsuitable for ovulation induction.

Effect of low temperatures on in-vitro matured bovine oocytes.

This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C or 0 degree C for 5, 10, or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P < 0.05) for oocytes maintained at 39 degrees C (73.2%) than for oocytes cooled at 20 degrees C (58.6%), 10 degrees C (47.3%), or 0 degree C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39 degrees C, 20 degrees C, 10 degrees C and 0 degree C, respectively (P < 0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10 degrees C (0.0%) or 0 degree C (0.9%), followed by 20 degrees C (7.1%) and 39 degrees C (16.5%; P < 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting > or = 2 pronuclei), there was no difference (P > 0.05) between oocytes cooled at 0 degree C (59.7%) or 10 degrees C (67.9%). However, penetration rates in these 2 groups were lower (P < 0.05) when compared to zona-free oocytes cooled at 20 degrees C (83.1%) or those maintained at 39 degrees C (83.1%). Zona-free oocytes had higher penetration rates (P < 0.05) when cooled at 0 degree C (59.7%) or 10 degrees C (67.9%) than zona-intact oocytes cooled at 0 degree C (37.3%) or 10 degrees C (47.2%). However, there was no difference in the penetration rate when zona-free and zona-intact oocytes were cooled at 20 degrees C or maintained at 39 degrees C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida.

Effects of low temperatures on in vitro produced bovine zygotes.

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C, or 0 degree C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0 degree C, 10 degrees C, 20 degrees C, and 39 degrees C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0 degree C or maintained at 39 degrees C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm.

Analysis of urinary progesterone metabolites with behavioral correlation in Guenther's dik-dik (Madoqua guentheri).

Urine samples and behavioral data were collected from nine adult female Guenther's dik-dik (Madoqua guentheri) from September 1989-November 1992. The durations of predefined individual behaviors were recorded during focal sampling periods and ad libitum. Immunoreactive pregnanediol-3-glucuronide (PdG) concentration in dik-dik urine was determined by radioimmunoassay. The immunoreactive PdG radioimmunoassay was validated for use with dik-dik by determining parallelism [F(alpha=0.05,1,4) = 0.04) between a serially diluted PdG standard and serially diluted dik-dik urine. Behavioral estrus was manifested by lordosis. Eighty-nine percent of the estrous behavior occurred during or within 2 days of the interluteal phase period, as delineated by immunoreactive PdG concentrations; thus, a high degree of correlation between hormone concentrations and behavioral data was observed. The mean (+/-SD) luteal phase, interluteal phase, and estrous cycle lengths were 14.4+/-5.5 days (range, 6-29 days; n = 50), 6.6+/-5.1 days (range, 2-33 days; n = 50), and 20.2+/-6.6 days (range, 10-43 days; n = 48), respectively. During the collection period, one animal conceived, with a gestation period of 170 days. Estrous cyclicity occurred throughout the year, with no evidence of seasonality. Cochromatography of pooled urine samples with radiolabeled PdG indicated that the major progesterone metabolite detected by the immunoreactive PdG antibody during luteal phase and pregnancy was not PdG. This is the first detailed description of female dik-dik reproductive endocrine activity.

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries ).

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.