Prevention of zona hardening in non-human primate oocytes cultured in protein-free medium.

BACKGROUND: Development of a protein-free medium for in vitro maturation of oocytes that prevents zona hardening is essential for the study of components that affect the maturation process. METHODS: Immature macaque oocytes were cultured in modified CMRL medium with serum protein or without protein [with or without polyvinyl alcohol (PVA)] for 24 hours. RESULTS: Sperm penetration of oocytes cultured for 24 hours prior to insemination was poor in CMRL medium alone, but was dramatically improved in CMRL medium with the addition of either PVA or BCS. In the second experiment, in vivo matured oocytes were cultured in CMRL with PVA or serum for 6 hours prior to insemination. The incidence of fertilization and embryo development to the blastocyst stage were similar in CMRL with PVA. CONCLUSIONS: These results indicate that fertilization failure occurs when macaque oocytes are cultured in medium without protein, but can be prevented with PVA.

Strategies for the production of genetically identical monkeys by embryo splitting.

Genetically identical rhesus monkeys would have tremendous utility as models for the study of human disease and would be particularly valuable for vaccine trials and tissue transplantation studies where immune function is important. While advances in nuclear transfer technology may someday enable monkeys to be cloned with some efficiency, embryo splitting may be a more realistic approach to creating pairs of genetically identical monkeys. Although several different approaches to embryo splitting, including blastocyst bisection and blastomere separation, have been used successfully in rodents and domestic species for production of pairs and sets of identical offspring, efforts to create monozygotic twins in rhesus monkeys using these approaches have not met with similar success. Aggregation of split embryos with other types of blastomeres, such as tetraploid and developmentally asynchronous blastomeres, that could potentially increase their cell numbers and developmental competence without contributing to term development has been investigated as an alternative approach to creating monozygotic twin monkeys. The major challenges encountered with respect to the efficient production of monozygotic twins in rhesus monkeys and potential strategies to overcome these challenges are discussed.

In vitro development and cell allocation following aggregation of split embryos with tetraploid or developmentally asynchronous blastomeres in rhesus monkeys.

Production of genetically identical pairs of monkeys would have tremendous implications for biomedical research, particularly immunological studies and vaccine trials. Specific aims of this study were to (1) determine whether aggregation of embryos split into halves or quarters with equal numbers of either developmentally asynchronous or tetraploid blastomeres would enhance their developmental potential in vitro and increase total cell numbers in resulting blastocysts, and (2) determine the allocation of tetraploid and developmentally asynchronous blastomeres in resulting blastocysts. Results demonstrated that development into blastocysts was greater (p < 0.05) for embryos split into pairs (39.8%) than for those split into quadruplet sets (17.4%) and similar (p > 0.05) to that of nonmanipulated controls (59.6%). Creation of chimeras from aggregation of a single 4-cell and four 16-cell stage blastomeres resulted in blastocyst formation (69.2%) similar to that of nonmanipulated control embryos (66.9%). However, neither development nor total cell numbers in resulting blastocysts differed between aggregate chimeras and those split into quadruplet sets at the 16-cell stage. Blastocysts resulting from the aggregate chimeras were derived strictly from the 16-cell stage blastomeres, with complete exclusion of the 4-cell stage blastomeres. Aggregation of split embryos with equal numbers of tetraploid blastomeres doubled (p < 0.05) both the proportion developing into blastocysts and the total cell numbers in resulting blastocysts. Tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm of resulting blastocysts. In conclusion, due to exclusion of the less advanced cells, aggregation of developmentally asynchronous blastomeres did not improve the developmental competence or cell numbers of split rhesus embryos. Reconstitution of split embryos with equal numbers of tetraploid blastomeres enhanced their developmental potential and cell numbers in resulting blastocysts. However, tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm.

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Birth of MHC-defined rhesus monkeys produced by assisted reproductive technology.

One of the best animal approaches for testing HIV vaccines is the challenge of vaccinated rhesus macaques with SHIV or SIV. Production of rhesus macaques in which all of the MHC class I and II alleles are known represents an opportunity to characterize the entire immune response to SIV and should be an invaluable resource for understanding pathogenesis and vaccine-induced immune responses. Unfortunately, there are few MHC-defined rhesus macaques available for vaccine research. Selective breeding supports the production of limited numbers of macaques that express particular MHC class I alleles. If both parents express the allele of interest, only three quarters of the offspring will express the same allele. However, assisted reproductive technologies, such as in vitro fertilization (IVF) and embryo transfer, can be used for production of MHC-defined macaques, expressing multiple MHC class I and II molecules for which SIV peptides, tetramers and ELISPOT assays exist. Here, we report the birth of MHC-defined rhesus monkeys produced by assisted reproductive technology. Continued improvements in assisted reproductive technologies in rhesus monkeys will enable us to develop a unique prototypic animal production program for the creation of MHC-defined and genetically-identical monkeys for vaccine research.

Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: short communication.

Although strategies for in-vitro maturation of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes, full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.

A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates.

A model to study mechanisms controlling nuclear and cytoplasmic maturation of primate oocytes is being developed in our laboratory. The high incidence of pregnancy failure in women following in-vitro fertilization (IVF) may be partly attributed to inadequate cytoplasmic maturation of oocytes. Advancement of knowledge of mechanisms controlling primate oocyte maturation would have important implications for treatment of human infertility, and would potentially increase numbers of viable non-human primate embryos for biomedical research. Use of a non-human primate model to study oocyte and embryo biology avoids legal, ethical and experimental limitations encountered in a clinical situation. Using this model, the meiotic and developmental capacity of oocytes from three sources have been compared: (i) in-vivo matured oocytes from monkeys stimulated with follicle-stimulating hormone (FSH) and human chorionic gonadotrophin, (ii) in-vitro matured oocytes from monkeys primed with FSH, and (iii) in-vitro matured oocytes from non-stimulated monkeys. This work demonstrates that oocyte developmental competence is likely acquired both during follicle development, before meiotic resumption, and during meiotic progression, concurrent with nuclear maturation. Potential causes of developmental failure of in-vitro matured oocytes, implications for human infertility, and future strategies to study the regulation of primate oocyte maturation are discussed.

Onset of nucleolar and extranucleolar transcription and expression of fibrillarin in macaque embryos developing in vitro.

Specific aims were to characterize the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during preimplantation development in vitro in macaque embryos using autoradiographic and immunocytochemical techniques. Autoradiography was performed on whole embryos cultured with [3H]uridine for assessment of nucleolar (rRNA) and extranucleolar (mRNA) transcription. Expression of fibrillarin was immunocytochemically assessed in whole embryos using a primary antibody against fibrillarin and a fluorescein isothiocyanate-conjugated secondary antibody. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos cultured 6-10 h with [3H]uridine. Culture with alpha-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with alpha-amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin.

Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium.

The formulation of chemically defined culture media that support primate embryo development would facilitate studies on primate preimplantation embryogenesis. The specific aims of this study were (i) to evaluate the development of the macaque embryos in a simple, chemically defined, protein-free medium developed for a rodent embryo model, and (ii) to determine if a two-step progressive culture system could enhance blastocyst development and zona escape. In experiment 1, in-vitro-fertilized pronucleate-stage embryos (n = 81) from nine monkeys were randomly allocated to one of three treatments: (a) hamster embryo culture medium-6 (HECM-6; chemically defined, protein-free medium), (b) CMRL-BCS medium (modified CMRL-1066 medium containing 20% bovine calf serum; BCS), and (c) a two-step culture procedure (HECM-6 through to the 8- to 12-cell stage, and CMRL-BCS medium beyond that stage). Optimal development was attained equally (P > or = 0.05) with embryos cultured in CMRL-BCS medium or the two-step procedure (48 and 61%), but not to the blastocysts respectively). HECM-6 alone supported development to the morula stage (72%) equally as well as CMRL-BCS medium (80%) or the two-step procedure (69%), but not to the blastocyst stage (22 versus 48 and 61% respectively). Hatching of the blastocysts was essentially limited to the serum-containing media (CMRL-BCS medium, 31%; two-step procedure, 44%). In experiment 2, in-vitro-fertilized pronucleate-stage embryos (n = 87) from nine monkeys were randomly placed in each of four two-step treatments: (a) HECM-6 through to the 8- to 12-cell stage and CMRL-BCS medium beyond that stage, (b) HECM-6 through the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HECM-6 through to the morula stage and CMRL-BCS medium beyond that stage, and (d) HECM-6 through to the morula stage and HECM-6-BCS beyond that stage. Greater (P < or = 0.05) percentages of embryos developed into blastocysts, expanded blastocysts and hatched blastocysts when switched at the 8- to 12-cell versus the morula stage in the second step medium. When transferred into BCS-containing medium at either the 8- to 12-cell or morula stage, embryos underwent blastulation and expansion equally well in CMRL-BCS medium versus HECM-6-BCS. However, when embryos were switched to the second step medium at the 8- to 12-cell stage, hatched blastocysts were obtained more (P < or = 0.05) frequently in CMRL-BCS medium (50.9%) than in HECM-6-BCS (37%). This work is the first to produce in-vitro-fertilized primate blastocysts cultured from the pronucleate stage in chemically defined, protein-free medium, and demonstrates that while primate embryos can form morulae in such a medium, their requirements for blastocoel formation and zona escape appear to be more demanding, and may be acquired as early as the 8-cell stage.

Granulosa cells from follicle stimulating hormone-primed monkeys enhance the development competence of in-vitro-matured oocytes from non-stimulated rhesus monkeys.

The specific aims of this study were to determine if culture with freshly recovered granulosa cells obtained from follicle stimulating hormone (FSH)-primed versus non-stimulated monkeys during in-vitro maturation (IVM) improves the meiotic and development capacity of oocytes from non-stimulated macaque monkeys and confers on them competence to develop into blastocysts in vitro. Antral follicles > or = 1 mm in diameter were dissected from the excised ovaries of 11 non-stimulated rhesus monkeys. Cumulus-enclosed germinal vesicle-stage oocytes (n = 282) were randomly cultured in each of three IVM treatments; (i) control (no granulosa cells), (ii) non-stimulated granulosa cells (5 x 10(6) live cells/ml) from non-stimulated monkeys, or (iii) FSH-primed granulosa cells (5 x 10(6) live cells/ml) from monkeys primed with purified FSH for 8.5 days. All treatments contained gonadotrophins (5 micrograms/ml FSH; 10 micrograms/ml luteinizing hormone). Oocytes were cultured for 36-41 h in 25 microliters drops (one to 10 oocytes per drop) of modified CMRL-1066 medium containing 20% bovine calf serum, and then inseminated and cultured in the same medium (minus gonadotrophins) until developmental arrest or zona escape. Nuclear maturation, fertilization and cleavage to the 8-cell stage did not differ for the three treatment methods. However, development to the 9- to 15-cell stage was improved (P < or = 0.01) by culture with either non-stimulated granulosa or FSH-primed granulosa cells compared with control. The culture of oocytes with FSH-primed granulosa but not non-stimulated granulosa cells enhanced (P < or = 0.05) development to the morula stage compared with control (23, 11 and 5% respectively). Oocytes cultured with FSH-primed granulosa cells were more (P < or = 0.05) competent to develop into blastocysts (6.5%) than controls (0.5%). In conclusion, the culture of immature oocytes from non-stimulated monkeys with fresh granulosa cells recovered from FSH-primed monkeys enhanced their competence to develop into morulae and blastocysts in vitro, and resulted in the first blastocysts produced in vitro from in-vitro-matured/in-vitro-fertilized oocytes of non-stimulated macaques.

Use of purified porcine follicle-stimulating hormone for ovarian stimulation of macaque monkeys.

At present, in nonhuman primates, ovarian stimulation with heterologous gonadotropin preparations is the only reliable way to produce substantial numbers of competent ova for in vitro fertilization and embryo development studies. Preparations such as equine chorionic gonadotropin (eCG) and human menopausal gonadotropins (hMG or hFSH) have been used successfully, but eCG is crude and contains variable amounts of LH activity, while hMG/hFSH is very expensive and the supply is not stable. This study examined the use of a purified porcine FSH preparation (Folltropin V) for ovarian stimulation in rhesus monkeys. Twice-daily intramuscular injections of this preparation resulted in good follicular development, and was followed by a single intramuscular injection of hCG. Ova were collected laparoscopically 30 h post hCG, fertilized in vitro and then cultured until development ceased. Stimulation of 9 monkeys with Folltropin V yielded a mean of 20 ova per animal, of which 71% reached metaphase II and were inseminated; of these, 92% were fertilized in vitro and 48% developed into blastocysts in vitro. These results are similar to those reported by us and by others using eCG, hMG or an hFSH/hMG combination for ovarian stimulation of macaque monkeys. We conclude that Folltropin V is a suitable alternative preparation for ovarian stimulation in nonhuman primates and one that also has the advantages of being readily available and much less expensive than human gonadotropin preparations.

Effects of granulosa cells and gonadotrophins on meiotic and developmental competence of oocytes in vitro in non-stimulated rhesus monkeys.

Specific aims of this study were to determine effects of granulosa cells and gonadotrophins on the meiotic and developmental competence in vitro of oocytes from non-stimulated rhesus monkeys. Oocytes (368) were obtained from small antral follicles (class 1: 700-999 microns; class 2: 1000-2500 microns) dissected from excised ovaries of 10 follicular and three luteal phase monkeys. Oocytes were cultured in one of four treatment groups: (i) granulosa cells (4 x 10(6) live cells/ml) + gonadotrophins (5 micrograms/ml follicle stimulating hormone and 10 micrograms/ml luteinizing hormone), (ii) granulosa cells alone, (iii) gonadotrophins alone, or (iv) controls (no granulosa cells or gonadotrophins). Mature oocytes were inseminated and cultured until arrest of embryo development. Meiotic and developmental capacity was greater (P < or = 0.05) for oocytes from class 2 compared with class 1 follicles and from luteal compared with follicular phase monkeys. Culture of oocytes with gonadotrophins alone during in-vitro maturation improved (P < or = 0.01) activation and cleavage through the 5-8-cell stage (46.5%). Culture with granulosa cells alone during in-vitro maturation augmented progression of embryos to the morula stage (6.3%). The combination of granulosa cells+gonadotrophins enhanced (P < or = 0.01) nuclear maturation, but had no effects on developmental capacity beyond those of either treatment alone. In conclusion, both granulosa cells and gonadotrophins during in-vitro maturation have stimulatory effects on the meiotic and developmental competence of oocytes from non-stimulated macaques in vitro.

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro.

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

Follicle-stimulating hormone priming of rhesus monkeys enhances meiotic and developmental competence of oocytes matured in vitro.

The objective of this study was to determine the effects of FSH priming of rhesus monkeys upon meiotic and developmental capacity of oocytes in vitro. FSH-primed monkeys (n = 9) received injections of porcine FSH for 6-7 days. Germinal vesicle-stage oocytes (n = 402) were obtained from dissected follicles. All oocytes were cultured for 36-40 h, inseminated, and incubated until developmental arrest. Greater (p < or = 0.001) percentages of cumulus-enclosed oocytes from FSH-primed vs. nonstimulated monkeys completed meiotic maturation (74 vs. 41%), activated/fertilized (85 vs. 61%), and cleaved from the 2-4-cell (79 vs. 38%) through the morula (29 vs. 1%) stages. Blastocysts (4%) were obtained only from FSH-primed monkeys. Compared with their cumulus-enclosed counterparts, denuded oocytes were compromised (p < or = 0.05) in completing nuclear maturation and cleaving in vitro. The meiotic and developmental capacity of denuded oocytes from FSH-primed monkeys exceeded (p < or = 0.01) those of denuded oocytes from nonstimulated monkeys. Denuded oocytes from FSH-primed monkeys were more (p < or = 0.01) competent to cleave beyond 8 cells than cumulus-enclosed oocytes from nonstimulated monkeys. Meiotic and developmental competence was similar between oocytes with or without cytoplasmic vesicles. Thus, FSH priming of monkeys enhances nuclear and cytoplasmic maturation of oocytes in vitro, and resulted in production of the first in vitro-matured/in vitro-fertilized primate blastocysts in vitro.

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro.

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 micrograms/ml human follicle-stimulating hormone (hFSH), (2) 10 micrograms/ml human luteinizing hormone (hLH), (3) 1.0 microgram/ml hFSH and 10 micrograms/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 +/- 1.7 hr; hLH: 10.1 +/- 1.8 hr; both gonadotropins: 8.8 +/- 1.1 hr) when compared to controls (17.4 +/- 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 +/- 1.2 hr; hLH: 35.1 +/- 1.4 hr; both gonadotropins: 35.2 +/- 1.3 hr; no gonadotropins: 34.1 +/- 1.2 hr). The mean interval to extrusion of PB1 was 34.1 +/- 0.6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys.

Specific aims of this study were to compare relationships between size of intact antral follicles and meiotic competence, diameters, and chromatin configurations of germinal vesicle (GV) oocytes in non-gonadotropin-stimulated rhesus monkeys. Intact antral follicles were dissected from excised ovaries of nine normally cycling monkeys in the follicular phase of the menstrual cycle and of two acyclic monkeys. Follicles were classified according to diameter: I (200-450 microns), II (451-700 microns), III (701-1000 microns) and IV (> 1000 microns). Cumulus-enclosed oocytes were released from follicles and either measured (diameter) and fixed immediately or cultured for 48 h in modified CMRL medium containing 0.5 micrograms/ml ovine FSH, 10 micrograms/ml ovine LH, and 10% bovine calf serum. Following Hoechst staining, three distinct patterns of chromatin organization (GV1-3) were identified in GV oocytes according to the degree of association with the nucleolar periphery (encapsulation or "rimming"). In antral follicles > 450 microns in diameter, perinucleolar encapsulation (GV3) of oocytes before culture and meiotic maturation (metaphase II) of oocytes after culture increased (p < 0.01) with antral follicle growth in a graded fashion. While 56.3% of oocytes from large (> 1000 microns) follicles completed maturation, few (9.3%) oocytes from small (200-450 microns) follicles were competent to mature in vitro. At 0 h of culture, class IV follicles contained a greater (p < 0.01) proportion of GV3 oocytes and a smaller (p < 0.01) proportion of GV1 oocytes than classes I, II, and III follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced postpartum anestrus of suckled beef cows treated with microencapsulated luteinizing hormone-releasing hormone analog.

This study evaluated the effect of microencapsulated LHRH agonist (D-Trp6-LHRH) on gonadotropin release and occurrence of estrus in early postpartum beef cows. Angus cows (n = 54) were assigned randomly to two treatment groups at d 5 postpartum. Group 1 received a single i.m. injection of D-Trp6-LHRH (LHRH-A) encapsulated in poly-DL-lactide-coglycolide, calculated to release 15 micrograms of LHRH-A per day for 30 d (n = 23). Group 2 received vehicle only (control, n = 31). Blood samples (15-min intervals for 6 h) were obtained on d 5, 10, 20, 30, and 40 postpartum for evaluation of LH and FSH concentrations (n = 12 per group). Days to first postpartum estrus were reduced by treatment with LHRH-A (Group 1, 43.7 +/- 4.2 d vs Group 2, 55.9 +/- 4.7 d; P < .05). However, days to conception were similar between groups (68.9 +/- 7.9 vs 76.7 +/- 6.7 d, respectively). On the day of treatment, cows treated with LHRH-A had higher mean concentrations of LH and FSH than did controls (8.3 +/- 1.4 vs 2.0 +/- .4 ng/mL for LH and 211.0 +/- 8.6 vs 51.2 +/- 2.7 ng/mL for FSH (P < .05). There were no differences in mean concentrations of LH or FSH between treatment groups on d 10, 20, 30, and 40 postpartum. Cows given LHRH-A had more (P < .05) LH pulses on d 10 and 30 postpartum than did controls. This study demonstrated that microencapsulated D-Trp6-LHRH reduced the postpartum anestrous interval in suckled beef cows.

Enclomiphene does not alter the postpartum interval of suckled beef cows.

The objective of this study was to determine whether an antiestrogen (enclomiphene) would shorten the interval to first estrus and conception in postpartum beef cows. Sixty postpartum Angus beef cows were stratified by age, body condition, and calving date and were randomly assigned to one of two treatment groups. Group 1 cows (n = 24) received three silastic implants, each containing 150 mg of enclomiphene, on d 20 postpartum. Implants were removed on d 30 postpartum. Group 2 cows (n = 28), received empty implants and served as controls. Cows were artificially inseminated at first detected estrus. Estrus detection and ovulation were further verified by increased serum progesterone. Concentrations and pulse frequencies of LH were determined from blood samples collected at 15-min intervals for 6 h on d 20, 25, 30, and 40 postpartum. Hypothalami and pituitaries were collected from four cows in each treatment group on d 30 postpartum and analyzed for concentrations of estradiol receptors. Concentrations of total and unoccupied hypothalamic and pituitary estradiol receptors were reduced by enclomiphene. Neither concentrations nor pulse frequencies of LH differed significantly between treatment groups on any of the 4 d. Days to first estrus did not differ (P greater than .05) between enclomiphene-treated (57 +/- 6; n = 24) and control (56 +/- 4; n = 28) cows. Days to conception did not differ between treated (81 +/- 9) and control (79 +/- 8) cows. The dose of enclomiphene used in this study reduced hypothalamic and pituitary estrogen receptors but did not alter secretion of LH or days to first estrus in the postpartum beef cow.

Phenotypic relationships of scrotal circumference to frame size and body weight in performance-tested bulls.

The objective of this study was to determine if frame size (height) is related to scrotal circumference. A total of 695 bulls (425 Angus, 65 Hereford, 70 Charolais, 135 Simmentals) were tested for postweaning gain in five stations over a period of 1 to 3 years. Variables examined included hip height, weight and scrotal circumference at beginning and completion of a 140-d feeding period. Correlations among these traits, adjusted for age (7 to 10 months at entry), year, station and management were estimated for each breed. Both height and weight were correlated positively with scrotal circumference at the start and the end of the test period in all four breeds. When height and scrotal circumference were adjusted for weight, correlations were negligible, with the exception of end-of-test values for Charolais bulls (-0.26). Negative correlations were obtained between the scrotal circumference at the start of the test and the change in height during the test after adjustment for weight in Angus bulls (-0.18) and in Charolais bulls (-0.15). These small negative phenotypic relationships indicate that a bull's fertility is not seriously reduced by large frame size at the completion of a feedlot performance test. For maximum fertility in bulls, scrotal circumference needs to be evaluated independently of frame size.