B.L. Dresser’s research while affiliated with Audubon Nature Institute and other places

Fishing cats (Prionailurus viverrinus) are small (6-15kg) spotted cats from dispersed areas of Southeast Asia found mostly in wetland habitats. They are classified by the International Union for Conservation of Nature (IUCN) as endangered, with a decreasing population, due to habitat loss and degradation. Few studies have been done on applying assisted breeding techniques to the species, although the birth of a live kitten after IVF/embryo transfer (ET) has been reported (2006 Theriogenology 66, 1518-1524). Here, we describe the birth of a live fishing cat kitten using the technique of laparoscopic intratubal AI. A ten-year-old female who had served previously as an oocyte donor (5×) following gonadotropin treatment was administered a total of 5IU of porcine FSH (Sioux Biochemical, Sioux City, Iowa) over 4 days (1×/day) followed by 10IU of porcine LH on the fifth day. At approximately 28h after LH treatment, the ovaries/oviduct were accessed by a laparoscopic technique comparable to that used for oviducal embryo transfer (ibid.). To deposit semen into the left oviduct, a 16-guage thin-wall trocar/needle was inserted into the abdominal cavity on the right side, approximately 1cm lateral to the midline and approximately 2 to 3cm below the umbilicus. A 14-cm open-end tom cat catheter was inserted into the 16-guage cannula (blunt) and the catheter tip was positioned underneath the fimbria overlaying the ovary. Then, a 50-mm length of 30-guage polytetrafluoroethylene (PTFE) thin-wall tubing containing approximately 30μL of freshly collected semen was threaded through the catheter and the sample was expelled with positive pressure from a threaded-plunger 1-mL syringe. The left ovary contained 7 to 8 preovulatory (3-4mm) follicles, 4 of which were manually ruptured immediately after deposition of semen with a 22-guage needle inserted through the 16-guage cannula. Then, with the 16-guage trocar/cannula in the same position (on the right side), the tip was redirected towards the right ovary and approximately 30μL of semen was deposited underneath the fimbria as described above. The right ovary presented with 5 to 6 preovulatory (3-4mm) follicles, 2 of which were punctured with the 22-guage needle after insemination. No ovulations were present on either ovary. The semen used for insemination was a fresh sample collected by electroejaculation from a 9-year-old male. The raw sperm concentration was 220 millionmL(-1), with 70% motility. The number of motile sperm deposited per oviduct was estimated to be approximately 4.6 million. The female was anesthetized 51 days later and radiography was done to determine her pregnancy status. A single fetus was present, so she was moved from an outdoor pen into a large indoor holding pen to allow for video-monitoring during the remainder of gestation. On Day 70, early signs of labour were observed and an elective Caesarean section was done approximately 4h later. A live, healthy male kitten weighing 204g was delivered. One year later, gonadotropin treatment/AI were repeated on the same pair. At approximately 30h post-LH treatment, preovulatory follicles were present, but fewer than the previous treatment (5-6 total). Two fresh ovulation sites were seen on the left ovary. Pregnancy was not established. A reason for the failure was not apparent, unless it was age related.

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.

The black-footed cat (Felis nigripes), a diminutive spotted cat whose native habitat is arid grasslands in South Africa, Namibia and Botswana, is classified as vulnerable by the International Union for Conservation of Nature and is listed as CITES Appendix I. They are perhaps the rarest of the African cats and their status is threatened by habitat deterioration and poisoning from ingestion of baited carcasses intended for other species of cats. Here, we examined (1) ovarian response of black-footed cat females to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF with cooled vs cryopreserved sperm and (3) in vivo developmental ability of in vitro-derived embryos. Six females, 1.5 to 2.5 years of age at first treatment, were administered a total of 3.0 to 3.6 IU of porcine FSH (IM; Sioux Biochemical Co., Sioux City, IA) daily over 4 days. On Day 5, 3.0 (n=12) or 5.0 (n=2) IU of porcine LH (IM; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24h later. One, two, three, or four LOR were done on 1, 3, 1 and 1 females, respectively (total=14 LOR). The average age at LOR was 3.3 years (range=1.5-7.5 years). Semen was obtained by electroejaculation of 3 males (1.5, 6.0, 7.5 years). Anaesthesia for LOR and electroejaculation was induced and maintained, after intubation, with 5 and 2.5% isoflurane, respectively. Sperm samples were used after storage at 4°C for 24h (TEST yolk, TY) or after cryopreservation (TY+6% glycerol). Luteal tissue was present on the ovaries at 4 of 5 LOR done during January to May as compared with none of 9 LOR done from June to December. Of 165 oocytes (mean=11.8) recovered, 38/54 (70%) and 50/106 (47%) underwent cleavage after IVF with cooled or cryopreserved sperm, respectively (P<0.01, chi-square). None of 5 oocytes cleaved after intracytoplasmic sperm injection with cryopreserved sperm. Procedures for in vitro embryo production were as described previously (Gómez et al. 2006 Theriogenology 66, 72-81; Pope et al. 2006 Theriogenology 66, 1518-1524). Four laparoscopic oviducal embryo transfer procedures were done on Day 1: 2 recipients received fresh Day 2 embryos (n=5, 8) and 2 recipients received embryos that had been cryopreserved on Day 1 (n=6) or 2 (n=8) at a slow, controlled rate in 1.4M of propylene glycol/0.125M of sucrose/10% dextran 70. Each recipient (1.75 to 4.5 years) had undergone LOR on Day 0 (5-19 oocytes recovered). Upon ultrasonographic examination on Day 50, a 2.3-year-old recipient of cryopreserved embryos was determined to be pregnant. She delivered 2 live male kittens, without assistance, on Day 69. When first examined at 15 days of age, the kittens weighed 156 and 198g. At 5 months, their weights were 1.62 and 1.81kg. The sperm sample used to produce the embryos (in 2005) that resulted in the births of kittens (in 2011) was collected from a male at the Henry Doorly Zoo, Omaha, NE (in 2003), extended and transported overnight at 4°C to New Orleans, LA, before cryopreservation. In summary, we have further demonstrated that assisted reproductive technology can be used for conservation of rare and vulnerable small felids.

Previously, we have shown that survival of cat sperm is maintained in both non-egg yolk, semi-defined extenders and in extenders with greatly reduced levels of egg yolk (2%). Usually, cryoprotectant is added to extended samples after gradual cooling to 4°C, but recent reports have shown that satisfactory sperm survival can be obtained after addition at 22°C. Here, our objectives were to examine sperm survival after (1) cryopreservation from 22°C vs after gradually cooling to 4°C or (2) cryopreservation in a completely defined extender without animal or plant proteins vs extender+2% egg yolk. Epididymides from local veterinary clinics were dissected in HEPES 199 medium (He199). The sperm suspension was filtered (40 μ), layered onto a density gradient column and centrifuged at 650×g for 20min. Then, the sperm pellet was resuspended in 1mL of He199 and centrifuged for 5min at 800×g and the subsequent pellet was extended in TEST Buffer with either 0% (0% EY) or 2% egg yolk (2% EY). Next, 0% EY samples were further split into 2 groups-either gradually cooled to 4°C before 12% glycerol (1:1) was added (4C-0%EY) or 12% glycerol (1:1) was added at 22°C without cooling (22C-0%EY). Control samples extended in 2% EY were cooled to 4°C before addition of 12% glycerol (1:1) (4C-2%EY). Samples were loaded into 0.25-mL straws and placed in a -80°C freezer for 20min before storage in LN2. Sperm samples were thawed in air (22°C) for 5s and immersed in a 60°C water bath for 5s. After a 7-step addition of He199, samples were centrifuged at 800×g for 5min and pellets resuspended in He199. Sperm samples were evaluated for motility (Mot; computer-assisted semen analysis, 37°C) at 0h (initial assessment), after cooling to 4°C (PC) and at 0-h (0-PT) and 3-h post-thaw (3-PT) incubation at 37°C. Membrane integrity (MI; SYBR 14-PI) and acrosomal status (AS; FITC-PNA) were analysed at the initial assessment, 0-PT and 3-PT. Results are shown in Table 1. At 4°C (PC), sperm extended in 0% EY and 2% EY maintained 92 and 91%, respectively, of their initial motility (66%). At 0-PT and 3-PT, motility in the 3 groups had decreased by >50% and >70%, respectively. Motility at 3-PT in the 22C-0%EY treatment was less than the other 2 treatments (P<0.05; 1-way ANOVA). At 0-PT, samples in the 4C-2%EY group had a higher membrane integrity value (P<0.05) than did the 22C-0%EY group, whereas that of the 4C-0%EY group was not different from the other 2 groups. However, at 3-PT, both groups cooled to 4°C before cryopreservation had higher membrane integrity values (P<0.05) than the group cryopreserved at 22°C. At 0-PT and 3-PT, the percentage of sperm with intact acrosomes ranged from 69% (4C-2%EY) to 59% (22C-0%EY) and from 55% (4C-2%EY) to 43% (22C-0%EY) of the initial value (89%), respectively. In summary, we demonstrated that cat epididymal sperm could be frozen successfully in a completely defined TEST-buffered extender. Furthermore, we confirmed that addition of cryoprotectant (i.e. glycerol) after gradual cooling to 4°C is beneficial to post-thaw survival.

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.

Abstract The black-footed cat (BFC; Felis nigripes), one of the smallest wild cats, is listed as threatened. Interspecies somatic cell nuclear transfer (Is-SCNT) offers the possibility of preserving endangered species. Development to term of interspecies BFC (Is-BFC) cloned embryos has not been obtained, possibly due to abnormal epigenetic reprogramming. Treatment of intraspecies cloned embryos with TSA improves nuclear reprogramming and in vitro and in vivo viability. In this study, we evaluated (1) whether covalent histone modifications differ between Is-BFC cloned embryos and their IVF counterparts, (2) the optimal TSA concentration and exposure times to modify the covalent histone patterns, (3) if TSA enhances in vitro and in vivo developmental competence of cloned embryos, and (4) expression of pluripotent genes. Results indicated that the covalent histone modifications of Is-BFC cloned embryos aberrantly differ from their DSH-IVF counterpart embryos. Aberrant epigenetic events may be due partially to the inability of the DSH cytoplasm to modify the restrictive epigenetic marks of the BFC nuclei after somatic cell nuclear transfer (SCNT). Incomplete remodeling of the histone H3K9me2 in Is-BFC cloned embryos possibly contributes to abnormal expression of pluripotent genes and low embryonic development. Treatment of Is-BFC cloned embryos with TSA remodeled the covalent pattern in H3K9ac and H3K9me2, resembling epigenetic patterns in IVF counterpart embryos, and resulted in activation of some pluripotent genes. However, genomic reprogramming of Is-BFC cloned blastocysts did not follow the same reprogramming pattern observed in DSH-IVF embryos, and in vitro and in vivo developmental competence was not enhanced.