Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5 °C.

Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with antioxidant activity. The diet of five stallions was supplemented with 20 g of Maca powder daily for a total of 60 days. A control group of five stallions received the same diet without Maca. Semen was collected once before the administration of Maca (D0), twice during the administration at 30 and 60 days (D30 and D60), and finally twice at 30 and 60 days after the end of the administration (D90 and D120). Ejaculates were processed for cooled shipping at 5 °C and evaluated in the laboratory for total and progressive motility, acrosome integrity, and lipid peroxidation after collection and after 24, 48, and 72 h of storage. Dietary supplementation with Maca improved sperm concentration (from 213 ± 80.4 to 447 ± 73.1 × 106 spz/mL) and total sperm count (from 10,880 ± 4377 to 24,783 ± 4419 × 106 spz). The beneficial effects of Maca supplementation on motility and acrosome integrity in the raw semen were detected from the end of treatment with Maca (D60) until the end of the study (D120). Furthermore, during cooling storage, total motility, progressive motility, and acrosome integrity declined more slowly in the Maca-treated group than in the control group. Lipid peroxidation did not change during cooling storage in either group and did not show a significant difference between the two groups. In this study, the dietary supplementation with Maca increased sperm production and stabilized semen quality during chilled storage.

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.

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.

The 450-band resolution G- and R-banded standard karyotype of the donkey (Equus asinus, 2n = 62).

Donkey chromosomes were earlier characterized separately by C-, G- and R-banding techniques. However, direct comparisons between G- and R-banding patterns have still not been carried out in this species. The present study reports this comparison at the 450-band level by using replication G- and R-banding patterns. Two sets of synchronized lymphocyte cultures were set up to obtain early (GBA+CBA-banding) and late (RBA-banding) BrdU incorporation. Slides were stained with acridine orange and observed under a fluorescence microscope. Reverse GBA+CBA- and RBA-banded karyotypes at the 450-band level were constructed. To verify G- and R-banding patterns in some acrocentric chromosomes, sequential GBA+CBA/Ag-NORs and RBA/Ag-NORs were also performed. The results of CBA-banding patterns obtained in 12 animals from 2 breeds showed a pronounced polymorphism of heterochromatin, especially in EAS1q-prox. Ideogrammatic representations of G- and R-banded karyotypes were constructed using only one common G- and R-banding nomenclature. In the present study both G- and R-banding patterns and relative ideograms are presented as standard karyotype for this species at the 450-band level.

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).

Analysis of STAT5A/AvaI gene polymorphism in four Italian cattle breeds.

The STAT5A/AvaI polymorphism was investigated with PCR-RFLP in a sample of 339 cattle belonging to four breeds: Italian Friesian, Jersey, Italian Brown, and Podolica reared in south Italy. All three possible genotypes for the C/T polymorphism were identified. In these breeds, PCR-RFLP showed the predominance of the TT genotype in Italian Brown and Jersey cows; in Podolica and Italian Friesian CT is the most frequent genotype. The frequency of the T allele ranged from 0.55 to 0.81 in the analyzed populations. The distribution of genotypic and allelic frequencies at this locus was significantly different among the four populations based on a chi2 test (P < 0.001), suggesting that the molecular characteristics of the STAT5A gene could be significantly affected by the breed selection. Gene heterozygosity, gene homozygosity, effective allele number, fixation index, and polymorphism information content (PIC) were calculated. The observed heterozygosity, as well as the Ne and PIC values, indicates high genetic variability in the Podolica breed. Podolica could be considered an interesting reservoir of genetic diversity for a species under high selective pressure elsewhere.

Sex chromosome abnormalities and sterility in river buffalo.

Thirteen male river buffaloes, 119 females with reproductive problems (which had reached reproductive age but had failed to become pregnant in the presence of bulls) and two male co-twins underwent both clinical and cytogenetic investigation. Clinical analyses performed by veterinary practitioners revealed normal body conformation and external genitalia for most females. However, some subjects showed some slight male traits such as large base horn circumference, prominent withers and tight pelvis. Rectal palpation revealed damage to internal sex adducts varying between atrophy of Mullerian ducts to complete lack of internal sex adducts (with closed vagina). All bulls had normal karyotypes at high resolution banding, while 25 animals (23 females and 2 male co-twins) (20.7%) with reproductive problems were found to carry the following sex chromosome abnormalities: X monosomy (2 females); X trisomy (1 female); sex reversal syndrome (2 females); and free-martinism (18 females and 2 males). All female carriers were sterile.

Chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis).

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.

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).

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.

Chromosome fragility in cattle with chronic enzootic haematuria.

Chronic enzootic haematuria (CEH) is a severe syndrome due to prolonged ingestion of toxic principles of bracken fern, such as quercetin and ptaquiloside. Little information is available on chromosomal instability of cattle with access to bracken fern and suffering from CEH. In the present study, 45 cattle, aged from 7 to 12 years and pastured in the south of Italy, were cytogenetically investigated for the first time in search of both chromosomal aberrations (aneuploidy, gaps, chromatid breaks, chromosome breaks and fragments) and sister chromatid exchanges (SCEs). Of these animals, 30 (group 1) had access to bracken fern and showed signs of CEH, and 15 (group 2; control) did not. Percentage of abnormal cells (aneuploidy, chromatid breaks, chromosome breaks and fragments) was higher in animals affected by CEH (34.7%, group 1) than that (24.3%) reached in the control (group 2). The same results were achieved when including gaps. Indeed, the mean number of cells with structural aberrations excluding gaps (chromatid breaks, chromosome breaks and fragments) per cell was higher (P<0.001) in animals affected by CEH (0.16+/-0.36) than that (0.09+/-0.29) found in the control. Chromosome fragility in cells of animals affected by CEH was also confirmed when applying the SCE test: statistically higher levels (P<0.001) of SCEs were observed in animals with CEH (7.35+/-3.59 SCE/cell, group 1) than those in the control (5.40+/-2.68 SCE/cell).

Increased frequencies of both chromosome abnormalities and SCEs in two sheep flocks exposed to high dioxin levels during pasturage.

During the past four years several livestock farms (sheep, cattle and river buffalo) in the provinces of Naples and Caserta (southern Italy) have been unable to sell their milk and other dairy products due to the levels of dioxins (17 different types) present in the milk mass exceeding the value permitted [3 pg/g of fat, as human WHO 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) equivalent]. While some farms, especially those showing relatively low levels of dioxins, have managed to reduce the dioxins in the milk below the permitted threshold by changing the diet, many, especially sheep farms, have failed to do so. Indeed, about 12,000 head of cattle, river buffaloes and sheep have so far been culled. In the present study, 34 and 42 sheep from two herds raised in the province of Naples (Acerra municipality) and where high levels of dioxins (50.65 and 39.51 pg/g of fat, respectively) were found in the milk mass, were cytogenetically investigated and compared with 20 sheep (control) raised 80 km away from the exposed area. Increases of both chromosome abnormalities (gap, chromosome and chromatid breaks) (17 and 8 times higher in the two exposed herds, respectively) and sister chromatid exchanges (SCEs) were found in both herds when compared with the control, and the differences were highly significant (P<0.001). No statistical differences were found when comparing the frequencies of aneuploid cells of exposed animals (16.4 and 17.8%) and control (17.9%). Furthermore, high levels of mortality and abnormal foetuses were recorded in one of the two farms when compared with the control.

Freemartinism in river buffalo: clinical and cytogenetic observations.

During the last five years forty phenotypic female river buffalos with reproductive problems and two phenotypic male co-twins of females, raised in the provinces of Caserta and Salerno (southern Italy) underwent cytogenetic investigation. Of the 42 animals studied, 10 freemartins (8 females and 2 males) were found with variable percentages of male and female blood cells, the majority however showing similar percentages of both. Of the eight females, six showed normal body conformation, vagina and clitoris, while two showed some male traits (tight pelvis). The two males were apparently normal with only a reduced size of one testicle in one animal. Clinical observations performed in the internal reproductive organs of the female carriers by both rectal palpation (5 females) and direct observation after mating (3 females) revealed serious damages varying from complete lack of internal sex adducts (closed vagina) to hypoplasia of Mullerian ducts and absence (or atrophy) of ovaries. All freemartin females were sterile. To our knowledge, this is the first detailed description of freemartinism in river buffalo by combining clinical and cytogenetic analyses.

Chromosome fragility in two sheep flocks exposed to dioxins during pasturage.

In the last 3 years several farms raising cattle, river buffalo and sheep have been unable to sell dairy milk due to the presence of high levels of dioxins. Furthermore, several cases of abortion (around 25% of total births) and abnormal foetuses (2.5% of total births) were recorded in two flocks of sheep raised in the province of Naples where a higher level of dioxins (5.27 pg/g fat, as human WHO TCDD equivalent) have been found in the milk mass than that permitted (3.0 pg/g fat, as human WHO TCDD equivalent). Cytogenetic investigations were carried out on 24 sheep (all females), randomly sampled from the two different flocks, one abnormal foetus and 11 female sheep (control) raised approximately 80 km from the area where the two exposed flocks were raised. Frequencies of aneuploid cells, gaps, chromatid breaks, chromosome breaks, fragments and sister chromatid exchange (SCE) were determined. While no differences were observed between the number of aneuploid cells (15% of total cell population) of both exposed animals and controls, significant (P < 0.001) increases in the frequencies of other chromosome abnormalities (mean chromosome abnormality/cell = 0.76 +/- 1.1) and SCEs (mean SCE/cell = 9.4 +/- 3.7) were found in the exposed animals, compared with the control (mean chromosome abnormality/cell = 0.18 +/- 0.4; mean SCE/cell = 7.1 +/- 3.0). Significantly higher values of SCEs (mean SCE/cell = 10.9 +/- 4.4) were also found in the abnormal foetus compared with the control. Chemical analyses on soil, grass and water at two sites where the two flocks were pastured established that doses of dioxins (17 different types) were below the legally permitted limits.