Invited review: Learning from the future-A vision for dairy farms and cows in 2067.

The world's population will reach 10.4 billion in 2067, with 81% residing in Africa or Asia. Arable land available for food production will decrease to 0.15 ha per person. Temperature will increase in tropical and temperate zones, especially in the Northern Hemisphere, and this will push growing seasons and dairy farming away from arid areas and into more northern latitudes. Dairy consumption will increase because it provides essential nutrients more efficiently than many other agricultural systems. Dairy farming will become modernized in developing countries and milk production per cow will increase, doubling in countries with advanced dairying systems. Profitability of dairy farms will be the key to their sustainability. Genetic improvements will include emphasis on the coding genome and associated noncoding epigenome of cattle, and on microbiomes of dairy cattle and farmsteads. Farm sizes will increase and there will be greater lateral integration of housing and management of dairy cattle of different ages and production stages. Integrated sensors, robotics, and automation will replace much of the manual labor on farms. Managing the epigenome and microbiome will become part of routine herd management. Innovations in dairy facilities will improve the health of cows and permit expression of natural behaviors. Herds will be viewed as superorganisms, and studies of herds as observational units will lead to improvements in productivity, health, and well-being of dairy cattle, and improve the agroecology and sustainability of dairy farms. Dairy farmers in 2067 will meet the world's needs for essential nutrients by adopting technologies and practices that provide improved cow health and longevity, profitable dairy farms, and sustainable agriculture.

Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles.

STUDY QUESTION: Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? SUMMARY ANSWER: AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. WHAT IS KNOWN ALREADY: In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. STUDY DESIGN, SIZE, DURATION: Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary follicles and their oocytes were also observed. These results were obtained only when AMH was added to cultures in advance of insulin (presumably because it penetrates tissue more slowly than insulin). Results of experiments with cortical pieces from fetal ovaries at mid-gestation, when follicles are forming, showed that AMH did not inhibit the formation of follicles. Immunohistochemical localization of AMH showed that it is not present in fetal ovaries until the third trimester, when it was localized to the granulosa cells of secondary and small antral follicles. LIMITATIONS REASONS FOR CAUTION: The experiments were performed with fetal ovaries because follicles form and follicle activation begins during fetal life in cattle (as it does in humans), so fetal ovarian cortex of later gestation provides tissue rich in primordial follicles. We assume, but have no experimental evidence, that our findings also apply to post-natal ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Although circulating AMH is used as an indication of the follicular reserve in women, little is known about AMH in human ovaries. Cattle are an excellent non-primate model for human ovarian follicular development and, hence, the findings suggest similar roles for AMH in human follicular development. LARGE SCALE DATA: Not applicable. STUDY FUNDNG/COMPETING INTEREST(S): This research was supported by National Research Initiative Competitive Grants no. 00-35203-9151, 2003-35203-13532, and 2008-35203-05989) from the U.S. Dept. of Agriculture's National Institute of Food and Agriculture to JEF and by an NIH National Research Service Award (F32 HD08264) to RAC. There are no conflicts of interest or competing interests.

DNA polymorphisms and transcript abundance of PRKAG2 and phosphorylated AMP-activated protein kinase in the rumen are associated with gain and feed intake in beef steers.

Beef steers with variation in feed efficiency phenotypes were evaluated previously on a high-density SNP panel. Ten markers from rs110125325-rs41652818 on bovine chromosome 4 were associated with average daily gain (ADG). To identify the gene(s) in this 1.2-Mb region responsible for variation in ADG, genotyping with 157 additional markers was performed. Several markers (n = 41) were nominally associated with ADG, and three of these, including the only marker to withstand Bonferroni correction, were located within the protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2) gene. An additional population of cross-bred steers (n = 406) was genotyped for validation. One marker located within the PRKAG2 loci approached a significant association with gain. To evaluate PRKAG2 for differences in transcript abundance, we measured expression in the liver, muscle, rumen and intestine from steers (n = 32) with extreme feed efficiency phenotypes collected over two seasons. No differences in PRKAG2 transcript abundance were detected in small intestine, liver or muscle. Correlation between gene expression level of PRKAG2 in rumen and average daily feed intake (ADFI) was detected in both seasons (P < 0.05); however, the direction differed by season. Lastly, we evaluated AMP-activated protein kinase (AMPK), of which PRKAG2 is a subunit, for differences among ADG and ADFI and found that the phosphorylated form of AMPK was associated with ADFI in the rumen. These data suggest that PRKAG2 and its mature protein, AMPK, are involved in feed efficiency traits in beef steers. This is the first evidence to suggest that rumen AMPK may be contributing to ADFI in cattle.

Influence of commercial inactivated or modified-live virus vaccination at time of AI on corpus luteum development and function in beef cattle.

Our objective was to evaluate the effect of vaccination with an inactivated virus vaccine (IVV) or modified-live virus (MLV) vaccine on the corpus luteum (CL). On d0, synchronized beef cows were treated with MLV (n = 70; BoviShield Gold FP5VL5), IVV (n = 16; ViraShield 6VL5HB), or were unvaccinated controls (n = 5). Plasma was collected from treated animals on d0 and every other day through d22. Plasma was analyzed for concentrations of progesterone and 15 cytokines. Between d10 and d13, selected females (n = 13) were ovariectomized; controls were slaughtered on d15/16 to obtain CL for histological evaluation. There were reduced numbers of large luteal cells (LLC) in MLV compared to IVV and controls (P < 0.0001), but IVV were similar to controls (P = 0.11). MLV had decreased LLC percentage compared to controls, and IVV were intermediate (P < 0.0001, MLV: 1.57 ± 0.33 %, IVV: 2.99 ± 0.30 %, Control: 6.45 ± 0.33 %). Based on progesterone concentrations, 24 % MLV and 0 % IVV had an abnormal cycle following vaccination. Overall, MLV had reduced progesterone concentrations (P = 0.02; MLV: 3.61 ± 0.22; IVV: 4.81 ± 0.46 ng/mL). The new CL that formed following an abnormal cycle in MLV had the greatest percentage (35.56 ± 5.5 %) of apoptotic cells. Treatment by cycle status interaction, and time significantly affected IFN-γ, IP-10, MIP-1ß, and MCP-1 (P < 0.03), with several time points having elevated concentrations in abnormally cycling MLV animals. Collectively, this demonstrates MLV vaccination around estrus negatively influenced LLC, progesterone, and increased luteal apoptosis and pro-inflammatory cytokines.

Role of the placenta in developmental programming: Observations from models using large animals.

Developmental programming, which proposes that "insults" or "stressors" during intrauterine or postnatal development can have not only immediate but also long-term consequences for healthy and productivity, has emerged as a major biological principle, and based on studies in many animal species also seems to be a universal phenomenon. In eutherians, the placenta appears to be programmed during its development, which has consequences for fetal growth and development throughout pregnancy, and likewise has long-term consequences for postnatal development, leading to programming of organ function of the offspring even into adulthood. This review summarizes our current understanding of the placenta's role in developmental programming, the mechanisms involved, and the challenges remaining.

RNA sequencing and iTRAQ proteomic data from an experiment examining the influence of conceptus presence and preovulatory estradiol on endometrial gene transcripts and proteins around maternal recognition of pregnancy in beef cattle.

RNA sequencing reads and isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data were used to determine the impact of conceptus presence and preovulatory estradiol concentration on function of the d16 uterus in beef cattle. Conceptuses and endometrial biopsies were collected from the uterine horn ipsilateral to the corpus luteum. Total cellular RNA was extracted from endometrium for RNA sequencing across two lanes of a NovaSeq S2, 2 × 50-bp run. Two independent uterine luminal fluid pools (ULF) were made for each group: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimensional liquid chromatography mass spectrometry. Transcript abundances were determined using DESeq2 (FDR <0.05, FC>2). Scaffold Q+ was used to quantitate peptide and protein identifications in ULF. Datasets include uterine transcript and protein abundances among highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. This information can be useful for further investigating the role of specific transcripts and proteins in the maintenance of early pregnancy in beef cattle. This dataset is related to the article 'Influence of conceptus presence and preovulatory estradiol exposure on uterine gene transcripts and proteins around maternal recognition of pregnancy in beef cattle' by E.J. Northrop-Albrecht, J.J.J. Rich, R.A. Cushman, R. Yao, X. Ge, G.A. Perry. Molecular and Cellular Endocrinology.

iTRAQ-Based proteomic dataset for bovine pre-ovulatory plasma and follicular fluid containing high and low Estradiol.

This is isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data on bovine plasma (PL) and follicular fluid (FF) containing high and low pre-ovulatory circulating concentration of estradiol (E2). The PL and FF were collected from nine beef cows that were identified to initiate a new follicular wave on day -4 during synchronization. Follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Estradiol concentrations in PL and FF were measured by radioimmunoassays. Plasma and FF were labeled as containing high E2 (PL HE2 and FF HE2) or low E2 (PL LE2 and FF LE2). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from the four PL and FF samples. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimentional liquid chromatography ESI-based mass spectrometry. Proteins were identified and quantified using SEQUESTTM search engine embedded in Proteome Discoverer. The proteins matched with at least one unique peptide at minimum 95% confidence were considered positive identifications. Protein expression levels were determined by assigned fold change of >2.0 or <0.5 between any pair from the four sample types. The paired comparisons made were PL HE2 and PL LE2, FF HE2 and FF LE2, PL HE2 and FF HE2, and PL LE2 and FF LE2. Protein Analysis Through Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to classify protein functions. This dataset includes the overview of workflow for identification and quantification of proteins and details on 231 proteins identified which includes 103 up- and down-regulate proteins. This dataset can be useful for further probing of the identified regulated proteins to better understand folliculogenesis and ovulation, particularly in bovine. This dataset is related to the article 'iTRAQ-Based Proteomic Analysis of Bovine Pre-ovulatory Plasma and Follicular Fluid' by P. A. Afedi, E. L. Larimore, R. A. Cushman, D. Raynie, G. A. Perry. Domestic Animal Endocrinology. https://doi.org/10.1016/j.domaniend.2021.106606.

iTRAQ-based proteomic analysis of bovine pre-ovulatory plasma and follicular fluid.

Bovine follicular fluid (FF) creates a unique microenvironment in follicles necessary for follicle growth, oocyte maturation, and estradiol (E2) production. The objective of this study was to analyze changes in proteins in FF and plasma (PL) from animals with high E2 (HE2) or low E2 (LE2) during the preovulatory period. Beef cows were synchronized, and follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Nine cows were selected and slaughtered, blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from both PL and FF. Peptides were labeled with iTRAQ reagents and quantified using 2-dimentional liquid chromatography ESI-based mass spectrometry. Estradiol was associated with protein changes in PL and FF. Protein expression changes between FF HE2 and FF LE2 were greater than between PL HE2 and PL LE2. There were 15 up-regulated proteins and 10 down-regulated proteins in FF HE2 compared to FF LE2, and 7 proteins up-regulated and 9 proteins down-regulated in PL HE2 compared to PL LE2. Several of the differentially expressed proteins function in follicle development and were mainly categorized under cellular process and metabolic process. Pathway analysis identified the up- and down-regulated proteins were predominantly associated with the complement and coagulation cascades. The data demonstrate E2 regulates a wide range of reproductive associated proteins in bovine PL and FF and can provide the basis for further investigation of specific processes involved in such regulation.

Review: Perspective on high-performing dairy cows and herds.

Milk and dairy products provide highly sustainable concentrations of essential amino acids and other required nutrients for humans; however, amount of milk currently produced per dairy cow globally is inadequate to meet future needs. Higher performing dairy cows and herds produce more milk with less environmental impact per kg than lower performing cows and herds. In 2018, 15.4% of the world's dairy cows produced 45.4% of the world's dairy cow milk, reflecting the global contribution of high-performing cows and herds. In high-performing herds, genomic evaluations are utilized for multiple trait selection, welfare is monitored by remote sensing, rations are formulated at micronutrient levels, health care is focused on prevention and reproduction is managed with precision. Higher performing herds require more inputs and generate more waste products per cow, thus innovations in environmental management on such farms are essential for lowering environmental impacts. Our focus is to provide perspectives on technologies and practices that contribute most to sustainable production of milk from high-performing dairy cows and herds.

Antral follicular count is a tool that may allow the selection of more precocious Bradford heifers at weaning.

Although antral follicle count is a repeatable parameter across life that is positively associated with fertility, its use at weaning as a tool to discard less fertile heifers has not been extensively evaluated. The hypotheses of this work are: 1) maximum antral follicle count (MAFC) is repeatable between weaning and pre breeding evaluations, allowing selection of more fertile heifers at an early age, 2) heifers with high MAFC have growth and development parameters linked to an earlier puberty and pregnancy, 3) MAFC has a positive correlation with AMH concentrations, so that both could be used inter changeably. In this study, Hereford (n = 42 and n = 50) and Braford (n = 40 and n = 50) females were used in years 1 and 2; respectively, in a completely randomized experimental design. Heifers were examined for five to ten days at two different moments (post weaning and pre service), to determine MAFC. The concentrations of Anti müllerian hormone (AMH) were evaluated on the day of MAFC assessment. Growth and development parameters were evaluated post weaning and pre service. The repeatability of MAFC between post weaning and pre service evaluations was poor in three cases (Hereford Year 1 = 0.36 and 2 = 0.39 and Braford, Year 2 = 0.32) but it was high for Braford in Year 2 (0.72). The AMH repeatability between post weaning and pre service evaluations was high in one case (Braford Year 2 = 0.72) and moderate in the others (Year 1, Hereford = 0.50 and Braford = 0.52 and Year 2, Hereford = 0.50). In Year 2, Braford heifers with greater MAFC attained puberty at an earlier age (r2 = 0.129; P = 0.0196). Also, diminished MAFC corresponded with decreased growth and development, thus less Braford heifers with low MAFC were inseminated (2/16), compared to those with medium (12/17) and high MAFC (7/17; P < 0.01). Moreover, Braford heifers with low AFC had less progesterone in the cycle post insemination but pregnancy rate was not affected. In Braford heifers in Year 2, there was a high correlation between MAFC and AMH concentrations (0.85 P < 0.001). The results of these experiments indicate that post weaning MAFC and AMH concentrations may be applied to select those Braford heifers that attain puberty at an early age, but these tools are not useful in Hereford heifers.

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function.

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n=37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6+/-0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5+/- 0.6 versus day 10 = 10.4 +/-0.6 primary follicles/section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.

Changes in ovarian function associated with circulating concentrations of estradiol before a GnRH-induced ovulation in beef cows.

These studies were conducted to evaluate causes for differences in circulating concentrations of estradiol before a GnRH-induced ovulation. Beef cows were synchronized by an injection of GnRH on day -7 and an injection of prostaglandin F2α (PGF2α) on day 0. In experiment 1, blood samples were collected every 3 h from PGF2α on day 0 to hour 33 after PGF2α and at slaughter (hour 36 to 42; n = 10). Cows were assigned to treatment group based on circulating concentrations of estradiol (E2): HighE2 vs LowE2. At slaughter, follicular fluid (FF) and granulosa cells were collected from the dominant follicle. In experiment 2, blood samples (n = 30) were collected every 8 h from PGF2α until the dominant follicle was aspirated via ultrasound-guided follicular aspiration to collect FF and granulosa cells (hour 38 to 46). In experiment 1, HighE2 had increased abundance of 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.02), and greater concentrations of estradiol and androstenedione (P ≤ 0.02) in the FF. In experiment 2, HighE2 had increased abundance of CYP11A1, 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase, and LHR (P ≤ 0.03) vs either LowE2 or GnRHLowE2. There was a tendency (P = 0.07) for LH pulse frequency to be increased in both the GnRHLowE2 and HighE2 compared with LowE2. HighE2 cows experienced increas in circulating concentrations of estradiol compared with LowE2. In conclusion, animals with greater concentrations of circulating estradiol before fixed-time AI experienced an upregulation of the steroidogenic pathway during the preovulatory period.

Estimates of epistatic and pleiotropic effects of () and () genetic markers on beef heifer performance traits enhanced by selection.

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC II) was subjected to marker assisted selection for 2 yr to equalize and genetic marker frequencies to evaluate the epistatic and pleiotropic effects of these markers on BW, reproduction, and first calf performance traits in replacement beef females ( = 171) managed under 2 postweaning development protocols. Traits evaluated on the heifers were birth BW, weaning BW, 11-mo BW, 12-mo BW, 13-mo BW, first breeding season pregnancy evaluation BW, first calving season BW, 11-mo puberty, 12-mo puberty, 13-mo puberty, first breeding season pregnancy, and first calf weaning rate. Additionally, heifer's first calf performance traits of ordinal calving date, first calf birth BW, and first calf weaning BW (with and without age adjustment) were analyzed. Selection to increase minor allele frequencies and balanced sampling across genotype classes enhanced the ability to detect all genetic effects except dominance × dominance epistasis. The × genotype effect was significant ( < 0.05) for 11-mo BW and 12-mo BW and tended to be significant ( = 0.08) for 13-mo BW. Consistently, for all 3 traits, the most significant effect among epistatic × genotype effects was the additive effect, with the G allele decreasing BW. There were no associations between × genotype and fertility related traits ( ≥ 0.46) in this study. Additionally, there were no × genotype associations with first progeny performance traits ( ≥ 0.14). The large effect of the additive × additive interaction on first calf weaning BW was imprecisely estimated, which may warrant further investigation.

Litter-of-origin trait effects on gilt development.

The preweaning litter environment of gilts can affect subsequent development. In a recent experiment designed to test the effects of diet on gilt development, litter-of-origin traits including individual birth weights, immunocrits (a measure of colostrum intake), sow parity, number weaned, and individual weaning weights were collected for approximately 1,200 gilts that were progeny of approximately 300 sows. Subsequently, BW, LM area, and backfat were measured at 100 d of age and at 28-d intervals until slaughter (260 d of age). From 160 d of age to slaughter, gilts were observed daily for estrus. At slaughter, the reproductive tract and 1 mammary gland were recovered. The reproductive tract was classified as cyclic or prepubertal; the number of corpora lutea was counted. Uterine horn lengths and ovarian dimensions were measured. Uterus and ovary samples from every 10th gilt were prepared for histological evaluation of uterine gland development and follicle counts, respectively. Mammary gland tissue protein and fat were assayed. Day of the estrous cycle at slaughter was calculated using the first day of the most recent standing estrus (d 0) recorded previous to slaughter. Each gilt development trait was analyzed for association with each litter-of-origin trait, after adjusting for dietary treatment effects. Uterine length, ovarian dimensions, mammary gland protein and fat, and uterine gland development were also adjusted for day of the estrous cycle at slaughter. All litter-of-origin traits were associated ( < 0.05) with growth traits. Top-down (backward elimination) multiple regression analysis indicated that BW and LM accretion in gilts was positively associated with immunocrit ( < 0.01), birth weight ( < 0.01), preweaning growth rate ( < 0.01), and parity ( < 0.01). Backfat accretion was positively associated with preweaning growth rate ( < 0.01), number weaned ( < 0.05), and parity ( < 0.05). Age at puberty was associated with birth weight (positive; < 0.01) and preweaning growth rate (negative; < 0.01). Total uterine length was positively associated with only birth weights ( < 0.05). Mammary gland protein was negatively associated with preweaning growth ( < 0.01). Mammary gland fat was positively associated with birth weight and number of piglets weaned ( > 0.05). These results indicate that colostrum consumption, birth weights, preweaning growth rate, number weaned, and parity are associated with gilt development traits during later life.

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.

Postweaning nutritional programming of ovarian development in beef heifers.

Peripubertal caloric restriction increases primordial follicle numbers at breeding, which may improve reproductive potential. Our hypothesis was that feed restriction was changing primordial follicle number through stimulation of follicle formation via leptin, roundabout axon guidance receptor, homolog 4 (), or or through inhibition of follicle activation via anti-Müllerian hormone (). Heifers ( = 30) were fed a ration consisting of 30% alfalfa hay, 69.8% corn silage, and 0.2% salt as DM. Heifers received the control diet for 42 d before an initial 6 heifers were ovariectomized at 8 mo of age. The remaining 24 heifers were divided into 2 treatment groups. Controls were offered 97.9 g DM/kg BW over the entire feeding period. Stair-step heifers received 67.4 g DM/kg BW for 84 d. Following the 84-d restriction, heifers were stepped up to receive 118.9 g DM/kg BW over a 15-d period and were held at this feeding level 68 d. At the end of the feed restriction (11 mo of age), ovaries were collected from 6 heifers per treatment, and at the end of the refeeding period (13 mo of age), ovaries were collected from 6 heifers per treatment. Plasma leptin concentrations were greater in control heifers than in stair-step heifers at 11 mo of age ( < 0.0001). In histological sections, stair-step heifers had more primordial follicles ( = 0.03) than control heifers at 13 mo of age. There was no difference in secondary or antral follicle numbers between dietary treatment groups or ages. Relative abundance of mRNA in ovarian cortex of control heifers was greater at 13 mo than at 11 mo or before feed restriction (8 mo; = 0.01). Relative abundance of mRNA in stair-step heifers at 13 mo was greater than before feed restriction ( = 0.02) and at 11 mo did not differ from 8 or 13 mo ( = 0.70). Relative abundance of mRNA in the ovarian cortex followed a similar pattern, being greater in stair-step heifers at 11 mo compared with control heifers ( = 0.001). At 13 mo, mRNA did not differ between treatments ( = 0.30). Abundance of mRNA in the ovarian cortex did not change due to dietary treatment or age ( > 0.10). In conclusion, developing heifers on a stair-step compensatory growth scheme resulted in larger ovarian reserve before the onset of breeding, which may have beneficial effects on increasing reproductive lifespan.

A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight.

The use of genetic markers to aid in selection decisions to improve carcass and growth characteristics is of great interest to the beef industry. However, it is important to examine potential antagonistic interactions with fertility in cows before widespread application of marker-assisted selection. The objective of the current experiment was to examine the influence of 2 commercially available markers currently in use for improving carcass traits, the myostatin (MSTN) F94L and µ-calpain (CAPN1) 316 and 4751 polymorphisms, on heifer development and reproductive performance. In Exp. 1, beef heifers (n = 146) were evaluated for growth and reproductive traits over a 3-yr period to determine if these polymorphisms influenced reproductive performance. In Exp. 2, heifers representing the 2 homozygous genotypes for the MSTN F94L polymorphism were slaughtered on d 4 of the estrous cycle and reproductive tracts were collected for morphological examination. In Exp. 1, there was a tendency (P = 0.06) for birth BW to be affected by MSTN with the Leu allele increasing birth BW in an additive fashion. Additionally, MSTN significantly affected the proportion of pubertal heifers by the start of the breeding season (P < 0.05) with the Leu allele additively decreasing the proportion pubertal; however, this did not result in a delay in conception or a decrease in pregnancy rates during the first breeding season (P > 0.15). The GT haplotype of CAPN1, which was previously associated with decreased meat tenderness, was associated with an additive decrease in birth BW of the first calf born to these heifers (P < 0.05). In Exp. 2, there were no differences between the MSTN genotypes for gross or histological morphology of the anterior pituitary, uterus, or ovaries (P > 0.05). From these results, we concluded that the MSTN F94L and CAPN1 polymorphisms can be used to improve carcass traits without compromising fertility in beef heifers. The influence of these markers on cow performance and herd life remains to be determined. While the delay in puberty associated with the MSTN F94L polymorphism did not negatively impact reproductive performance in heifers, caution should be used when combining this marker with other markers for growth or carcass traits until the potential interactions are more clearly understood.

Age at puberty, ovulation rate, and uterine length of developing gilts fed two lysine and three metabolizable energy concentrations from 100 to 260 d of age.

The objective of this study was to determine the effect of ad libitum feeding diets differing in standard ileal digestible (SID) lysine and ME concentrations that bracket those fed to developing gilts in U.S. commercial settings. Average SID lysine and ME concentrations in diets currently fed to developing gilts were obtained from a poll of the U.S. commercial swine industry. Crossbred Large White × Landrace gilts (n = 1,221), housed in groups, were randomly allotted to 6 corn-soybean diets in a 2 × 3 factorial arrangement formulated to provided 2 SID lysine and 3 ME concentrations. Gilts received grower diets formulated to provide 1.02% (control = survey average) or 0.86% (control minus 15%) SID lysine and 2.94, 3.25, or 3.57 (survey average ME ± 10%) Mcal of ME/kg from 100 d of age until approximately 90 kg BW. Then, gilts were fed finisher diet containing 0.85% (control = survey average) or 0.73% (control minus 15%) SID lysine and 2.94, 3.26, or 3.59 (control ± 10%) Mcal of ME/kg until 260 d of age. Gilts were weighed, and backfat thickness and loin muscle area were recorded at the beginning of the trial and then every 28 d. Starting at 160 d of age, gilts were exposed daily to vasectomized boars and observed for behavioral estrus. At approximately 260 d of age, gilts were slaughtered and their reproductive tract was collected. Each reproductive tract was examined to determine whether the gilt was cyclic, the stage of estrus cycle, ovulation rate, and uterine length. Data were evaluated for normality and analyzed using mixed model methods. Average age at puberty was 193 d of age with a range from 160 to 265 d. When all gilts on trial at 160 d of age were included in the analysis, 91.0% reached puberty as determine by observation of standing estrus. Differences between dietary treatments on age at puberty or measurements of the reproductive tract were not detected. Growth rates to 160 d were not limiting for attainment of puberty in response to daily boar stimulation from 160 d.

The primordial to primary follicle transition.

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. A better understanding of the signals that initiate follicular growth in mammals, and of the conditions necessary for sustained growth of early preantral follicles in vitro, could have practical implications for contraception, alleviation of infertility, and regulation of the rate of follicle depletion (menopause). Our laboratory has developed two experimental systems that can be used to study factors involved in the activation of primordial follicles. In the first experimental system, small pieces of ovarian cortex, containing mostly primordial follicles, are isolated from fetal ovaries of cattle or baboons and cultured in serum-free medium. Under these conditions most primordial follicles become activated between 12 and 24 h of culture; their granulosa cells change shape, from flattened to cuboidal, and begin to express proliferating cell nuclear antigen (PCNA). During 7 days in culture, the newly-formed primary follicles and their oocytes increase significantly in diameter. This wholesale 'spontaneous' activation in serum-free medium is quite different from the much more gradual exit of primordial follicles from the resting pool that occurs in vivo and suggests that primordial follicles in vivo may be subject to a tonic inhibition of growth initiation or, alternatively, that some aspect(s) of the environment in vitro stimulates growth initiation. Recently we developed a second experimental system for studying activation of primordial follicles. Pieces of ovarian cortex from bovine or baboon fetuses were grafted beneath the developing chorioallantoic membrane (CAM) of 6-day-old chick embryos, a site known to support xenografted tissues. The cortical pieces were rapidly vascularized and histological analysis of pieces recovered after 2, 4, 7, or 10 days 'in ovo' revealed no increase in the number of primary follicles and maintenance of original numbers of primordial follicles. Therefore, grafting ovarian cortical pieces beneath the chick CAM provides an experimental system in which follicles remain at the primordial stage in a readily accessible environment and which, thus, may be used to study potential regulators of the initiation of follicle growth. The results suggest that vascularization of isolated pieces of ovarian cortex provides conditions that maintain follicular quiescence, whereas culture in vitro allows unrestrained activation of primordial follicles. Future studies with and comparisons of the in vitro and in ovo models may provide new insight into the mechanisms that regulate the primordial to primary follicle transition.

Effect of long-term treatment with recombinant bovine somatotropin and estradiol on hormone concentrations and ovulatory response of superovulated cattle.

The objective was to assess effects of long-term treatment with recombinant bovine somatotropin (bST) and estradiol-17beta (E2) on the number of follicles that ovulated in response to FSH. Non-lactating Holstein and Jersey cows (Trial 1, n=27) and Angus cows and heifers (Trial 2, n=35) received two ear implants of E2 and biweekly injections of bST in a 2 x 2 arrangement of treatments. Estradiol implants were removed 74.6 +/- 1.1 d after insertion and 18.1 +/- 0.9 d after the last biweekly injection of bST. Cows were stimulated with FSH-P beginning 2 d after removal of E2 implants, and PGF2alpha (PGF) was given on the third day of FSH treatment. Ovaries were collected to determine the number of CL at 1 to 2 wk after treatment with PGF. In Trial 2 only, cattle were inseminated at estrus and embryos were collected 6 to 8 d later. Implants of E2 increased (P < 0.01) serum E2 8-fold initially and E2 was still elevated 5-fold at removal of implants. Injections of bST increased (P < 0.01) serum growth hormone (GH) 15-fold and insulin-like growth factor-I (IGF-I) 3-fold. In Trial 1, number of CL was increased by the combination of bST+E2 (P < 0.01). In Trial 2, E2 increased the number of CL (P < 0.05), and bST increased the number of total ova and transferable embryos (P < 0.01). We conclude that long-term treatment with bST and E2 may interact to enhance follicular development and ovulatory response to FSH.