Sara Albarella

Effect of energy source during culture on in vitro embryo development, resistance to cryopreservation and sex ratio

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.

XX/XY Chimerism in Cattle: Clinical and Cytogenetic Studies

The freemartin condition represents the most frequent form of intersexuality found in cattle, and occasionally in other species. Freemartinism arises when vascular connections occur between placentae of developing heterosexual twin foetuses, XX/XY chimerism develops, and ultimately there is masculinisation of the female tubular reproductive tract to varying degrees. The aim of this work was to report the clinical and cytogenetic studies performed in 28 cattle co-twins, 24 of which were cytogenetically chimeric (2n = 60, XX/XY), raised in the region of Campania (southern Italy). Clinical findings of the 16 freemartin females examined varied greatly, from a more female phenotype (normal body conformation with the presence of a blind-ending vagina and primordial ovarian and uterus structures) to a nearly male phenotype (body conformations with male traits and presence of primordial prepuce, penis and testicles). The 8 freemartin males, in spite of the presence of XX cells, had a normal body conformation and external genitalia and some of them were fertile. In addition to cytogenetic diagnosis we also verified chromosome fragility by testing for chromosome aberration (CA: aneuploidy, gaps, chromatid breaks, chromosome breaks and fragments) and sister chromatid exchange (SCE). Freemartins showed a higher percentage of aneuploid cells and significant statistical differences in mean values of gaps, chromatid breaks and chromosome breaks when compared with control animals. To our knowledge, this is the first time that chromosome instability has been evaluated by analyses of CA and SCE in freemartin cattle.

Increased SCE levels in Mediterranean Italian buffaloes affected by limb malformation (transversal hemimelia)

In recent years some buffalo farms in Campania have reported the birth of calves with limb malformation, especially with transversal hemimelia. We investigated 20 Mediterranean Italian buffaloes (8 males and 12 females) from one day to six months of age, of which 10 were affected by transversal hemimelia (group 1) and 10 were healthy controls (group 2). The following clinical and radiological patterns were observed in the malformed animals: hind limbs amputated, the right amputated off the second tarsus bones and the left amputated off the proximal epiphysis metatarsus, and the right thoracic limb hypoplasic (1 female); left hind limb amputated off the proximal epiphysis metatarsus (2 females and 1 male); left hind limb amputated off the third tarsus bones (1 female); left hind limb amputated off the tibia (1 female and 1 male); left hind limb amputated off the distal epiphysis metatarsus (1 female); left hind limb amputated off the first phalanx (1 male); right hind limb amputated off the proximal epiphysis metatarsus (1 male). In their malformed limbs all the animals presented more or less developed outlines of claws. The mean rate of SCE/cell in animals with transversal hemimelia was 8.80 ± 3.19, that of the controls 6.61 ± 2.73. The difference was statistically significant (P < 0.001).

Chromosome instability in Mediterranean Italian buffaloes affected by limb malformation (transversal hemimelia).

For several years, a genetic disease called transversal hemimelia (TH), also known as congenital amputation, has been spreading in Mediterranean Italian buffalo. TH is characterized by the lack of limb distal structures, normally developing proximally to the malformed limb and being amputated at different points distally. A sample of 13 animals affected by TH was examined using the chromosome aberration (CA) test to better characterize chromosome instability already emerging in a preliminary study where we found a significantly higher difference (P < 0.001) in the mean rate of sister chromatid exchange/cell (8.80 +/- 3.19) performed in 10 malformed animals, when compared with the control (6.61 +/- 2.73). The percentage of aneuploid cells was higher in animals with TH (12.76) than in control animals (7.85). Mean gaps are greater in cells of animals with TH (6.62 +/- 2.38) than those found in the control (2.86 +/- 1.01) and similar results were obtained in chromatid breaks (0.13 +/- 0.31 and 0.07 +/- 0.06, respectively), chromosome breaks (0.11 +/- 0.27 and 0.06 +/- 0.13, respectively) and CAs excluding gaps (0.24 +/- 0.47 and 0.13 +/- 0.18, respectively). All these differences are statistically highly significant (P < 0.001).

Molecular and Cytogenetic Studies in a Case of XX SRY-Negative Sex Reversal in an Arabian Horse

An 18-month-old Arabian foal characterized by a stallion-like appearance was submitted for cytogenetic and molecular genetics examinations due to abnormalities of external genitalia and the presence of ovotestis-like structures in the abdominal cavity. By RB-banding the animal showed the normal female equine karyotype (2n = 64,XX). Molecular analysis revealed the absence of the SRY and ZFY genes and the presence of ZFX, a typical female equine condition. The entire RSPO1 coding region was examined to exclude its involvement. Although a SNP was found in exon 3, it was not responsible for an amino acid substitution.

Native cattle breeds of Southern Italy: karyological profile

Abstract Italian typical products of animal origin are strictly linked to native breeds. Their protection requires control of their reproductive and productive abilities. Hence the need for karyological studies to identify subjects with chromosome abnormalities linked to hypofertility or sterility. We report the results of karyological analyses carried out from January 2008 to December 2008 on 145 cattle of native breeds (Agerolese, Cinisara, Modicana and Podolica) reared in Southern Italy so as to evaluate and characterize the presence of chromosome abnormalities in subjects with normal phenotypes. Besides the 128 karyologically normal subjects (2n=60, XY and 2n=60, XX), 17 were carriers of rob (1;29) and one male was a carrier of cellular chimerism 2n=60, XX/XY. According to our data there is a high frequency of rob (1;29) in Cinisara and Podolica breeds while in Agerolese there was only one case of rob (1;29) and none in Modicana.

Elucidating the genetic basis of an oligogenic birth defect using whole genome sequence data in a non-model organism, Bubalus bubalis

Recent strong selection for dairy traits in water buffalo has been associated with higher levels of inbreeding, leading to an increase in the prevalence of genetic diseases such as transverse hemimelia (TH), a congenital developmental abnormality characterized by absence of a variable distal portion of the hindlimbs. Limited genomic resources available for water buffalo required an original approach to identify genetic variants associated with the disease. The genomes of 4 bilateral and 7 unilateral affected cases and 14 controls were sequenced. A concordance analysis of SNPs and INDELs requiring homozygosity unique to all unilateral and bilateral cases revealed two genes, WNT7A and SMARCA4, known to play a role in embryonic hindlimb development. Additionally, SNP alleles in NOTCH1 and RARB were homozygous exclusively in the bilateral cases, suggesting an oligogenic mode of inheritance. Homozygosity mapping by whole genome de novo assembly also supported oligogenic inheritance; implicating 13 genes involved in hindlimb development in bilateral cases and 11 in unilateral cases. A genome-wide association study (GWAS) predicted additional modifier genes. Although our data show a complex inheritance of TH, we predict that homozygous variants in WNT7A and SMARCA4 are necessary for expression of TH and selection against these variants should eradicate TH.

A rare case of simple syndactyly in a puppy

A case of non-syndromic, complete syndactyly involving all four limbs is described in a three-month-old male crossbreed dog for the first time. Syndactyly is a rare condition in most animal species, in dogs it has been infrequently reported. Findings of clinical, radiographic and cytogenetic analyses are described and demonstrate probably for the first time that numerical and structural chromosome aberrations are not involved in the pathogenesis of this case of syndactyly.

A new translocation t(1p;18) in an Italian Mediterranean river buffalo (Bubalus bubalis, 2n = 50) bull: cytogenetic, fertility and inheritance studies.

In recent years increasing attention has been paid to the cytogenetic control of Italian Mediterranean river buffalo (BBU) bulls authorized as sires which are registered in the stud book. Chromosome abnormalities described in this species are mainly numerical and affecting sex chromosomes. During routine cytogenetic analyses performed on young Italian Mediterranean river buffalo bulls in the progeny test, 1 animal was found to be carrier of a never before reported translocation t(1p;18) originated by fission of BBU1 and subsequent centric fusion of BBU1p with BBU18 as demonstrated by both R-banding and FISH-mapping techniques using specific molecular markers of BBU1p (DEFB1) and BBU18 (GPI). According to sperm analyses the semen characteristics were in physiological ranges, but the calf crop percentage was only 48.77% instead of 70–80%. Cytogenetic analyses performed on 50 offspring (36 females and 14 males) showed that 15 of them (30%) were carriers of the same translocation.

Diagnosis of XX/XY Blood Cell Chimerism at a Low Percentage in Horses

&NA; Disorders of sexual development (DSDs) are common in horses and cause economic loss in horse breeding. Thus, it is important to develop methods for unambiguous and fast identification of affected horses shortly after birth, as well as those that may propagate the condition to the next generation. Genetic causes of DSDs are multivarious and still little known, and thus development of diagnostic tests requires accumulating knowledge about individual cases and their etiologies. In particular, it is necessary to perform clinical, ultrasound, surgical, histological, cytogenetic, and genetic analyses with close attention in all the affected individuals. This report describes the case of a XX/XY chimeric horse with reproductive apparatus abnormalities and a very low percentage of XY cell in blood highlighting that to avoid undiagnosed case of cell chimeras, above all when studying DSD cases, it is essential to perform both genetic and cytogenetic analyses possibly on more than one tissue. HighlightsDisorders of sexual development (DSDs) are still a main cause of economic loss in horse breeding.Genetic and cytogenetic analyses on various tissues are essential in horse DSDs.DNA profiling on blood alone is not enough to detect all cases of cell chimerism.