Elisha Gootwine

Litter-size-dependent intrauterine growth restriction in sheep

Regulation of foetal development in sheep depends on interactions between the intrinsic capacity of the foetus for growth and the maternal environment. Lambs born in multi-foetus litters have relatively small placentae with fewer cotelydons, and lower birth weights. Litter-size-dependent intrauterine growth restriction (IUGR) is evident at mid gestation when metabolic needs of the conceptus are moderate, and overnutrition of ewes with multiple foetuses does not promote growth of their foetuses to the size of singletons. Those observations suggest that placental and conceptus growth in multi-foetus pregnancies is reprogrammed at mid gestation by an as yet undefined mechanism to attenuate foetal growth. This may protect the foetus from severe nutritional insult during late gestation, when its daily growth rate is at a maximum. In that way, lambs born in large litters with relatively lower birth weights may not experience the long-term physiological insults that can be observed in small lambs born to undernourished ewes.

Lamb and milk production of Awassi and East-Friesian sheep and their crosses under Mediterranean environment

Abstract Data on lambs born per ewe put to the ram (LB/EP), lambs born per ewe lambing (LB/EL), milk production through lactation and lactation length up to six lambings of 603 Awassi (A), East-Friesian (EF), A × EF (F1), F1 × F1 (F2), EF × F1 (1/4A), 1/4A × F1 (3/8A1) and 3/8A1 × 3/8A1 (3/8A2) ewes bred in the same flock in the years 1956–1971 were analysed. The data were obtained from 2293 ewe-years, 1993 lambings and 1698 lactations. Genotype, age at lambing and sire within genotype had an (P

Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia

Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

A mutation in gene CNGA3 is associated with day blindness in sheep

Lambs with congenital day blindness show diminished cone function, which is characteristic of achromatopsia, a congenital disorder described in humans and dogs. To identify gene(s) associated with sheep day blindness, we investigated mutations in the CNGA3, CNGB3, and GNAT2 genes which have been associated with achromatopsia. Sequencing the coding regions of those genes from four affected and eight non-affected lambs showed that all affected lambs were homozygous for a mutation in the CNGA3 gene that changes amino acid R236 to a stop codon. By PCR-RFLP-based testing, homozygosity for the stop codon mutation was detected in another 19 affected lambs. Non-affected individuals (n=386) were non-carriers or heterozygous for the mutation. While a selection program has been launched to eradicate the day blindness mutation from Improved Awassi flocks, a breeding nucleus of day-blind sheep has been established to serve as animal models for studying human achromatopsia.

Carrying the FecB (Booroola) mutation is associated with lower birth weight and slower post-weaning growth rate for lambs, as well as a lighter mature bodyweight for ewes

The present study was conducted in an Assaf flock in which the FecB (Booroola) mutation was segregated to determine whether the FecB mutation affects birthweight and the pre- and post-weaning growth rate of ewe lambs, as well as the mature bodyweight of ewes. Significant differences (P = 0.01) in birthweight (mean +/- s.e.m.) were found between BB ewe lambs (4.03 +/- 0.08 kg) and B+ and ++ ewe lambs (4.16 +/- 0.04 and 4.32 +/- 0.07 kg, respectively), which themselves did not differ significantly (P > 0.05). An FecB-associated maternal effect on the birthweight of ewe lambs was also detected, with the birthweight of lambs born to BB mothers (3.93 +/- 0.08 kg) being significantly (P < 0.0001) different from the birthweight of lambs born to B+ and ++ mothers (4.26 +/- 0.04 and 4.33 +/- 0.07 kg, respectively), which did not differ significantly. The genotypes of the lambs did not affect their preweaning growth rate. However, the post-weaning growth rate of ewe BB lambs (274 +/- 5 g day(-1)) was significantly (P = 0.05) different from the similar (P > 0.05) post-weaning growth rates of B+ and ++ lambs (284 +/- 3 and 290 +/- 4 g day(-1), respectively). The genotype at the FecB locus also affected the mature bodyweight of ewes, with that of BB ewes (67.3 +/- 1.4 kg) being significantly (P < 0.001) different from the similar mature bodyweight of B+ and ++ ewes (70.8 +/- 1.1 and 70.1 +/- 1.7 kg, respectively).

Reproductive performance and milk production of the improved Awassi breed as compared with its crosses with the Booroola Merino

Ovulation rate, embryo survival, lamb production, lamb survival and milk production of Awassi and BooroolaAwassi crossbred ewes, kept indoors, were compared. Awassi were non-carriers while Booroola × Awassi (F 1 ) and about half of 3/4 Awassi-1/4 Booroola (BQ) ewes were heterozygous at the FecB gene. Mean ovulation rate increased by 1·5 to 1·6 corpora lutea per ewe ovulating and prolificacy by 0·7 lambs born per ewe lambing in Fj and BC 1 (B+) ewes as compared with Awassi. Embryo survival rates in BC 1 ewes with two, three and four ovulations were 0·83, 0·68 and 0·71, respectively. Lamb survival rates at 1 day of age were 0·93, 0·90 and 0·77 and average birth weight was 4·9, 4·0 and 3·0 kg for lambs born as singles, twins and triplets, respectively. Average milk production of the Awassi was 506 I per ewe per lactation. F 1 and BC 1 ewes produced respectively, proportionately 0·48 and 0·63 of the Awassi milk production and there was no significant difference in milk production between BC 1 ,(B+) and BC 1 (++) ewes. The relatively low milk production of the Booroola Awassi crosses suggests that heterosis and recombination effects on milk production were negative. It is concluded that incorporation of the B allele per se can increase lamb production in the Awassi without affecting its milk production.

Active immunization of ewes against ovine placental lactogen increases birth weight of lambs and milk production with no adverse effect on conception rate.

In two experiments, 16 Booroola-Assaf and 35 Assaf ewe-lambs were actively immunized at 5 months of age against recombinant ovine placental lactogen (oPL). At 9 months of age, the ewe-lambs were mated for the first time and then introduced into a frequent mating-system. Anti-oPL antibody titers, reproductive performance, maternal serum levels of oPL during pregnancy, lamb birth weight and milk production of the ewes were followed in the immunized ewes and in their non-immunized control counterparts. All the immunized ewes developed anti-oPL antibodies, which interfered with oPL bioactivity in an in vitro cell proliferation assay. Conception rates did not differ (P>0.05) between immunized and non-immunized ewes. Abundant antibody-bound non-active oPL detected in sera of immunized ewes by western blotting indicated enhanced oPL production by the placenta following immunization. An increase (P<0.02) in serum oPL bioactivity, but not immunoreactivity, was observed in the immunized ewes in late gestation relative to control ewes. The average litter size was 1.83 and 1.32 lambs born per ewe lambing in the first and second experiments, respectively. Average birth weights of lambs born to the immunized ewes were higher (P<0.01) than for lambs born to control ewes by 10, 17 and 39% for those born as singles, twins and triplets, respectively. Immunized ewes produced 19 and 33% more milk (P<0.02) than the control ewes in the first 3.5 months of the first and second lactations, respectively. These findings do not suggest a role for oPL in maternal recognition of pregnancy, but they strongly suggest important roles for oPL in fetal growth and mammogenesis. Immunization of ewes against oPL may thus represent a novel practical technique for enhancing birth weights of lambs born to prolific sheep, as well as milk production by both dairy and mutton ewes.

A novel day blindness in sheep: epidemiological, behavioural, electrophysiological and histopathological studies.

Four genetically related Improved Awassi sheep flocks had sporadic births of lambs with congenital visual impairments that differed from other known forms of sheep blindness. Pedigree analysis suggested an autosomal recessive mode of inheritance. Behavioural studies of 4-month old affected lambs showed that their day vision (but not night vision) was impaired. Electrophysiological results at this age demonstrated diminished function of cones but not rods. Histopathological and immunohistochemical evaluation of affected retinas from 5-month old lambs revealed both red-green and blue cones, suggesting that the behavioural day blindness and reduced cone electroretinograms reflect cone dysfunction rather than severe cone photoreceptor loss. Awassi day blindness may be a form of achromatopsia.

Variability in the rate of decline in birth weight as litter size increases in sheep

Carrying multiple foetuses leads to a decline in lamb birth weight. The rate of litter size-dependent birth weight decline (LSDBD) in a population can be obtained by calculating the linear relationship between the reciprocal of lambs birth weight and the respective litter size. Based on published data on lamb birth weight and by using the reciprocal approach, LSDBD rate was calculated for 70 purebred and crossbred sheep populations in which birth weight of lambs born as singles ranged from 1·3 to 6·3 kg. High variability in LSDBD was found. Both genetic and environmental factors may contribute to the variability of this trait.

Ovine growth hormone gene duplication—structural and evolutionary implications

The growth hormone (GH) gene belongs to a gene family that also includes the chorionic somatomammotropin (placental lactogen) gene, the prolactin gene, and several prolactin-like genes, all evolved through series of gene duplications (Ohta 1993; Wallis 1993, 1994, 1996). The ovine GH gene is about 1.8 kb long and contains five exons and four introns (Byrne et al. 1987; Orian et al. 1988). Previous studies (Valinsky et al. 1990; Gootwine et al. 1993) have shown that gene duplication occurred at the ovine GH locus, and two alleles are found: the GH1 allele with a single GH copy, and the GH2 allele with two gene copies designated GH2-N and GH2-Z. The GH2 allele is found in wild sheep (Valinsky et al. 1990) and is the more frequent allele in most of the domesticated sheep breeds studied (E. Gootwine, unpublished results), suggesting that carrying the duplicated GH2 allele may have a selective advantage. It is not yet clear whether the GH2-N and the GH2-Z gene copies are similar in their structure or whether they code for different peptides. In sheep carrying the duplicated GH2 allele, only the GH2-N copy is expressed in the pituitary (Gootwine et al., 1996). Recently, it was found that GH is expressed also in the ovine placenta (Lacroix et al. 1996) where three GH mRNA variants were found. The relationship between these GH variants and the GH1, GH2-N, and GH2-Z is not yet clear. In the present study we looked at the nucleotide sequences of each of the GH1, GH2-N, and GH2-Z copies of the ovine GH and compared them with DNA sequences of pituitary and placental oGH expressed genes. Genomic DNA samples were obtained from an Awassi ewe and a Romney ewe homozygous for the GH1 allele, and from an Awassi ewe and a Romanov ewe homozygous for the GH2 allele. The sheep were selected from breeds that are unrelated and which were separated early in sheep domestication. The Awassi is a fat-tailed Asiatic breed, the Romanov belongs to the north European short-tailed breeds, and the Romney originated from the southeast England Romney Marsh breed. Regions of the GH1 gene copy were amplified from DNA extracted from GH1/GH1 sheep by means of the PCR reaction as described previously (Gootwine et al. 1993), with sets of primers (Table 1, Fig. 1A) that were designed on the basis of the published sequence of the oGH gene (Byrne et al. 1987; Orian et al. 1988). Regions of the GH2-N and GH2-Z copies were amplified from DNA extracted from GH2/GH2 sheep, after the DNA was fractionated. Genomic DNA fractions containing either the GH2-N or the GH2-Z copies were obtained by HindIII digestion: 150 mg of DNA from a homozygous GH2/GH2 sheep was digested with HindIII (Boehringer Mannheim, Germany) and separated on an 0.8% (wt/vol) agarose gel. Gel slices containing 8.3-kb DNA fragments that included the GH2-N copy only, or 5.8-kb DNA fragments that included the GH2-Z copy only, were excised, and the DNA was electroeluted and ethanol precipitated. DNA for sequencing was prepared from PCR products after purification with DS Primer Remover (Adv. Gen. Tech. Corp., Gathersburg, MD, USA). Direct PCR cycle sequencing was performed with the original amplification primers and dye termination labeling in an Applied Biosystems Inc. Model 373A automated DNA sequencer. Templates were sequenced from both ends. The full length of the GH genes of the GH1/GH1 Awassi and Romney ewes, 80% of the lengths of the GH2-N and GH2-Z gene copies of the GH2/GH2 Awassi ewe, and 53% and 76% lengths respectively, of the GH2-N and GH2-Z gene copies of the GH2/ GH2 Romanov ewe were obtained (GeneBank Accession numbers AF002110-AF002129; Fig. 1B). The structure of the two GH1 gene copies was identical and exactly matched (100%) the previously published oGH sequence (Orian et al. 1988), which will be considered here as a reference sequence. The structure of the GH2-N copy of the Awassi ewe was also identical in its coding region to the reference sequence, while the sequence of the coding region of the GH2-N copy of the Romanov sheep differed at one position, predicting histidine instead of asparagine at amino acid No. 148 of the hormone molecule. Two to eight nucleotide differences were found at the noncoding regions between the GH2-N sequences and the reference sequence. Five substitutions were found in the coding region of the Awassi GH2-Z gene copy compared with the reference sequence. Three were nonsynonymous substitutions leading to the presence of leucine, arginine, and serine instead of proline, glycine, and glycine in amino acid positions Nos. −7, 9, and 63, respectively. Two sequence differences leading to the same amino acid substiCorrespondence to: E. Gootwine