Male mule foal qualifies as the offspring of a female mule and jack donkey

Whereas mules are normally considered sterile, reports of fertile female mules persist. However, none subjected to cytogenetical and blood typing analyses could be confirmed as having indeed been mules that transmitted maternal markers to their alleged offspring. Reported here is the case of a cytogenetically verified female mule whose alleged male foal qualifies as offspring by a male donkey. The male foal is karyotypically a mule.     

Sex ratios in mule duck embryos at various stages of incubation

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.     

Characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis

A red cell antigen of donkeys and mules was identified using antibodies in serum from a mare which produced a mule foal affected with neonatal isoerythrolysis (NI). Subsequently antibodies with similar activity were identified in the sera of other mares which had produced mule foals and were produced by immunization of horses with blood from donkeys. The antigen detected by these antibodies does not correspond to any recognized horse red cell alloantigen. This may be a xenoantigen since all donkeys (and mules) tested have shared this antigen and all horses tested have lacked the antigen. The results suggest that all mule pregnancies (donkey sire x horse dam) are incompatible with regard to this factor and a potential for neonatal isoerythrolysis exists in all cases.     

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.     

Isolation and chemical composition of the leucocytes from donkey, horse, mule and pig.

1. Leucocytes from the blood of adult and young donkeys (Equus asinus L.), adult horses (Equus caballus L.), adult mules (Equus asinus x Equus caballus) and adult pigs (Sus scrofa L.) were obtained in a high degree of purity (99.9%) using Na2-EDTA-dextrans mixtures. 2. Sialic acids were released, purified, identified and determined from both non-delipided and delipided leucocytes. 3. N-glycolylneuraminic was the predominant sialic acid. N-acetylneuraminic acid and N,O-diacetyl-neuraminic acid were also found in all materials. Except in pig, other unidentified sialic acid(s) were also detected. 4. The concentration of total sialic acids (microgram/mg protein) is different according to the species, and in donkey species according to the age. 5. Galactose, glucose, mannose, fucose and (in a less amount) ribose were determined. Their total content is about 2-3-fold that of hexosamines. 6. There is a higher cholesterol content in adult donkey leucocytes than in those of young ones. 7. Total lipids, cholesterol or phospholipid contents are similar among the leucocytes of the above-mentioned species. 8. The similarities are marked in the electrophoresis patterns of proteins and glycoproteins for the donkey, mule and horse samples. 9. The molecular weights for leucocytes proteins were estimated as ranging between 230,000 and 20,000; and for the main protein bands, between 120,000 and 22,000.     

Eleven generations of selection for the duration of fertility in the intergeneric crossbreeding of ducks

A 12-generation selection experiment involving a selected line (S) and a control line (C) has been conducted since 1992 with the aim of increasing the number of fertile eggs laid by the Brown Tsaiya duck after a single artificial insemination (AI) with pooled Muscovy semen. On average, 28.9% of the females and 17.05% of the males were selected. The selection responses and the predicted responses showed similar trends. The average predicted genetic responses per generation in genetic standard deviation units were 0.40 for the number of fertile eggs, 0.45 for the maximum duration of fertility, and 0.32 for the number of hatched mule ducklings'' traits. The fertility rates for days 2–8 after AI were 89.14% in the S line and 61.46% in the C line. Embryo viability was not impaired by this selection. The largest increase in fertility rate per day after a single AI was observed from d5 to d11. In G12, the fertility rate in the selected line was 91% at d2, 94% at d3, 92% at days 3 and 4 then decreased to 81% at d8, 75% at d9, 58% at d10 and 42% at d11. In contrast, the fertility rate in the control line showed an abrupt decrease from d4 (74%). The same tendencies were observed for the evolution of hatchability according to the egg set rates. It was concluded that selection for the number of fertile eggs after a single AI with pooled Muscovy semen could effectively increase the duration of the fertile period in ducks and that research should now be focused on ways to improve the viability of the hybrid mule duck embryo.     

Ribosomal RNA expression in a mammalian hybrid,the hinny

The expression of nucleolus organizer region (NOR) activity in diploid cells was investigated in a model mammalian hybrid system, the hinny (female ass x male horse), by sequential Ag-NOR and chromomycin A3/distamycin A/DAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes. In the majority of cases we found non-expression of the horse-derived NOR chromosomes in the hinny. However, in one case there was strong NOR expression on horse-derived chromosome no. 1.     

Analysis of mechanisms regulating the expression of parental alleles at the GPD locus in mule erythrocytes

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was examined by 13% starch gel electrophoresis in 74 mules (42 females and 32 males), 35 donkeys, and ten horses. The quantitative expression of the parental alleles at the Gpd locus varies greatly in female mules from the hemizygous expression of the maternal allele to that of the paternal. The data obtained indicate that the X chromosomes are randomly inactivated in female mules. No selective advantage of a cell population with a maternally (or paternally) derived X active was found in female mule erythrocytes. It is suggested that the phenotypic variability in the expression of the parental Gpd alleles is related to the random proportions established between cells having either a maternal or paternal X active in an initiator (stem) cell group giving rise to erythroid tissue. Initiator cell numbers estimated for erythroid tissue (six or seven) are close to those reported for human females and intergeneric fox hybrids. These numbers may vary depending on the duration of the time of determination and the division rate of initiator cells at determination.     

Detecting population structure and recent demographic history in endangered livestock breeds: the case of the Italian autochthonous donkeys

Since its domestication, about 5000 years ago, the donkey (Equus asinus) has been extensively used as a work or draft animal in agricultural activities and for the transportation of people and goods. In the last century, technology improvement and growing mechanization strongly affected agriculture and the management and use of this livestock species in the industrialized countries. Nowadays, the use of donkeys for work or transport has almost disappeared, together with the need for mules or hinny breeding. During the last five decades, Italian autochthonous donkey populations suffered from a severe reduction in population size, which led to the extinction of several breeds. At present, eight breeds remain, all classified by FAO as critically endangered or endangered: Asinara, Pantesco, Grigio Siciliano, Romagnolo, Amiatino, Sardo Grigio, Martina Franca, and Ragusano. To evaluate the extant genetic variability of Italian donkeys, we typed 16 microsatellite loci in 258 individuals from these breeds. The results highlighted moderate levels of inbreeding ( F _IS = 0.127) and a significant partition of genetic variation into breeds, as suggested by fixation index ( F _ST = 0.109) and analysis of molecular variance (10.86% of total variation assigned to the between‐breeds level) analyses. This was confirmed by a Bayesian clustering procedure that also highlighted a further partitioning at lower hierarchical levels corresponding to the farms of origin. This evidence suggests that an effective management strategy for Italian donkey populations should focus on breeds as conservation units. However, this requires a synergic management strategy at the farm level to maintain diversity and avoid inbreeding.     

Does overfeeding enhance genotype effects on liver ability for lipogenesis and lipid secretion in ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on liver ability for lipogenesis and lipid secretion in ducks. Samples of liver and blood were collected at 14 weeks of age from 8 birds per group. Plasma levels of insulin was considerably increased in overfed ducks (1.9-fold), stimulating the hepatic activity of the main enzymes involved in lipogenesis from glucose (glucokinase, GK, glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX), while cytochrome-c oxidase (COX) activity, indicating overall oxidation ability of energy-yielding substrates, remained unchanged. Plasma levels of triglycerides, phospholipids and total cholesterol were therefore increased (1.9, 3.7, 1.6 and 1.6-fold, respectively). Glycaemia also significantly increased (+8%). Pekin ducks exhibited higher levels of GK and G6PDH activity in the liver than Muscovy ducks, suggesting a greater ability to use glucose consistent with their lower glycaemia. Muscovy ducks had greater ACX activity, suggesting greater ability to synthesise lipids. However, plasma lipid levels were much higher in Pekin ducks than in Muscovy ducks, suggesting a greater ability to export lipids from the liver. Values for the different criteria measured in this study were intermediate or similar in hinny and mule ducks to those of parental species. The high values for GK, G6PDH, ME and ACX activity in hybrid ducks enabled them to produce heavy fatty livers with the same chemical and lipid composition as Muscovy ducks and characterised by high amounts of triglycerides (around 96% of total lipids), and saturated and mono-unsaturated fatty acids.     

Gene expression in an interspecific hybrid: Analysis of hemoglobins in donkey,horse, and mule by peptide mapping

The fast and slow components of horse, donkey, and mule hemoglobin were analyzed by mapping of tryptic peptides. The two horse components share a common beta chain but differ in the alpha chain. Donkey hemoglobin differs from the slow horse component by replacement of histidine by asparagine in position 20 and replacement of a glycine residue by alanine in peptide V. Mapping of mule hemoglobins indicates that (1) the slow mule component contains the alpha and the beta chains of the slow horse component and the alpha and beta chains of the donkey, and (2) the fast mule component contains the alpha chain of the fast horse component and the beta chains of both the horse and the donkey. It is concluded that all six theoretically possible tetrameric combinations of the polypeptide chains encoded by the horse and donkey parental genes are formed in the mule.     

Does overfeeding enhance genotype effects on energy metabolism and lipid deposition in breast muscle of ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on energy metabolism and lipid deposition in breast muscle of ducks. Samples of breast muscle (Pectoralis major) were collected at 14 weeks of age from 8 birds per group. Overfeeding induced an accumulation of lipids in breast muscle (1.5- to 1.7-fold, depending on genotype) mainly induced by triglyceride deposition. It also induced a considerable increase in the amounts (expressed as g/100 g of tissue) of saturated and mono-unsaturated fatty acids (SFA, MUFA), while the amounts of poly-unsaturated fatty acids (PUFA) remained unchanged in hinny and Muscovy ducks or slightly increased in Pekin and mule ducks. In breast muscle, overfeeding decreased the activity of the main enzymes involved in lipogenesis from glucose (glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX). Lipoprotein lipase (LPL) activity in Pectoralis major muscle was also significantly decreased (-21%). The ability of muscle tissues to catabolize long-chain fatty acids, as assessed by beta-hydroxyacyl CoA dehydrogenase (HAD) activity, was increased in Pectoralis major muscle, as was cytochrome-c oxidase (COX) activity. Hybrid and Pekin ducks exhibited higher levels of ACX and LPL activity in Pectoralis major muscle than Muscovy ducks, suggesting a greater ability to synthesise lipids in situ, and to take up circulating lipids. Total lipid content in breast muscle of hybrid and Pekin ducks was higher than in that of Muscovy ducks. In hybrid and Pekin ducks, lipid composition of breast muscle was characterized by higher amounts of triglycerides, SFA and MUFA than in Muscovy ducks. Finally, oxidative metabolism was greater in Pectoralis major muscles of hybrid and Pekin ducks than in Muscovy ducks, suggesting an adaptative strategy of muscle energy metabolism according to lipid level.     

Characterization of a partial male fertility derived from Ogura-cms in progeny of a resynthesized Brassica napus

Summary The inheritance of a partial male fertile phenotype in somatic hybrid B. napus plants that carried novel mtDNA was investigated over five backcross generations to B. napus Triton. The recurrent parent and the original somatic hybrid both contained chloroplasts resistant to atrazine. The F₁ population contained mainly plants that were partial fertile, and some of the plants differed in mtDNA. The partial fertility predominated in the progeny of each backcross generation, but fully male sterile and fertile plants were also obtained. However, the sterility/fertility of these latter plants was not stable; both the fully male sterile and the male fertile plants produced progeny that were again predominantly partial male fertile. This pattern of predominant partial fertility but occasional sterile and fertile plants persisted in different nuclear backgrounds. Neither the male sterility nor the male fertility could be fixed and made stable. Test crosses indicated that restorer genes were probably not associated with appearance of male fertile plants. The evidence indicates that the behavior of the partial male fertility is cytoplasmic, and probably controlled by the chondriome.     

Chromosome studies of a donkey-grevy zebra hybrid

Summary Leukocyte and skin cultures of a female hybrid between a donkey and Grevy zebra show a diploid chromosome complement of 48. From this finding it is inferred that the Grevy zebra is characterized by 2n=34 and that such hybrid's regular infertility is analogous to that of mules and hinnies.Barr bodies were present in the tissue-cultured cells and studies with tritiated thymidine show that Barr body and the chromosome set replicate asynchronously. One large element corresponds to the late replicating X of other species and often possesses the morphologic features of a donkey X. At other times it is decidedly more metacentric, a quality tentatively assigned to the Grevy zebra X. Not enough cells could be studied to determine whether random or selective late replication (? inactivation) of one or the other of the sex chromosomes takes place. It is suggested that the study of such hybrids may yield cytologic information concerning the lyon hypothesis.Supported by a Grant from the USPH Service, GM 10210.The authors acknowledge gratefully the assistance of Mrs. Jane Juergens in photographic work.     

Genetic-cytoplasmic male sterility in progeny of 4x-2x crosses in cultivated potatoes

Summary Forty-three percent of the progeny from 4x × 2x crosses [Group Tuberosum cultivar x diploid hybrid (Groups Phureja or Stenotomum x Group Tuberosum haploid)] were male sterile. In contrast only four percent of the progeny from the reciprocal crosses were male sterile. Male sterility among the former progeny is presumed to result from the interaction of Tuberosum cytoplasm with dominant genes from the cultivated diploids, Groups Phureja and Stenotomum, an interaction known to occur in crosses of Tuberosum haploid x cultivated diploid species (2n = 2x = 24). The frequency of fertile progeny from the 4x × 2x crosses (57%) was significantly higher than that from the 2x × 2x crosses (Tuberosum haploid x cultivated diploid), (28%). The frequency of male fertility among progeny from different cultivars in 4x × 2x crosses varied from 31–82 percent. The difference between cultivars strongly suggests that some cultivars may have dominant male fertility restorer genes.     

Fertility in an F1 male hybrid of white-tailed deer (Odocoileus virginianus) x mule deer (O. hemionus)

The fecundity of an F1 male hybrid deer, from a cross between a male Odocoileus virginianus (white-tailed deer) and a female O. hemionus (mule deer), was assessed by cytogenetic and flow cytometric techniques. Analysis of chromosome morphology, nucleolus organizer expression, meiotic chromosome pairing, sperm production, and nuclear gene inheritance revealed no genetic anomalies that could potentially impair normal fertility. These observations are discussed in relation to recent reports of hybridization between natural populations of these two species.     

10种杜鹃亚属植物种间杂交的可育性研究

为探索杜鹃花亚属内异种杂交的可育性及其规律,对杜鹃亚属有鳞大花亚组(subsect. Maddenia)、三花杜鹃亚组(subsect. Triflora)、亮鳞杜鹃亚组(subsect. Heliolepida)及腋花杜鹃亚组(subsect. Scabrifolia)等4亚组10个杜鹃花种类的22个杂交组合(其中18个数据完整组合)的可育性进行了研究。结果表明:(1)所涉及的杜鹃亚属不同亚组间及三花杜鹃亚组内杂交均较困难,在18个数据完整组合中高可育与可育组合比率明显偏低,不育比率高(55.6%)。(2)在10个不可育或败育组合中,不能坐果(Cab型)、不能结实(Sab型)和可结实而种子不能发芽(Sng型)的数量分布为6∶1∶3,其不亲和或败育发生的阶段可能涵盖了从前合子期到后合子期的整个阶段。(3)亲本一方为多倍体组合的可育率(41.6%),尤其是母本为多倍体时,比二倍体组合的可育率(50.0%)低且无高可育组合出现,部分印证了倍性是导致该亚属植物不同种类杂交不亲和、不育与育性下降的重要原因,但不是唯一原因,亚组间杂交的可育率(16.7%)明显低于亚组内(三花杜鹃亚组内,58.3%)。(4)与相应的母本自然授粉结果相比,杂交明显导致多数可育组合绿苗率比率和单位可育种子数量比率的大幅度下降,这是双亲遗传差异及多倍体亲本介入后所导致的杂交衰退hybrid weakness现象。(5)在多倍体作母本的情况下,杂交单向不育或非对称遗传渗透现象明显。     

Masculinization mechanism of hybrids in bitterlings (Teleostei: Cyprinidae)

The sex ratio of bitterling hybrids (subfamily: Acheilognathinae) is often likely to be biased toward males. Artificial hybridization was carried out in 10 species of bitterlings (three genera) in order to elucidate the masculinization mechanism of hybrids. Tanakia himantegus never produced viable F1 hybrids with other species, while hybrids of most other species were viable. In terms of sex ratio and fertility, hybrids were clearly divided into two groups: congeneric Tanakia hybrids and others. Both male and female congeneric Tanakia hybrids were fertile. The sex ratio was nearly 1:1 in all groups of Tanakia hybrids. Except for the congeneric Tanakia hybrids, sterile males appeared predominantly in groups of hybrids in which females were very rare but remained fertile. Sterile intersexes were also observed in five hybrid groups: T. lanceolata (female) x Acheilognathus cyanostigma (male), Rhodeus uyekii (female) x T. lanceolata (male), A. rhombeus (female) x T. lanceolata (male), A. rhombeus (female) x T. limbata (male), and A. tabira tabira (female) x A. cyanostigma (male). In the development of male-predominant hybrids, although hybrid and control (parental species) hatching and survival rates do not differ, no females appeared in hybrids, contrary to the controls. Taking the female heterogametic sex-determining system (ZW) and the phylogenetic relationship of bitterlings into consideration, the masculinization mechanism of hybrids in bitterlings can be explained by the interaction of two sex chromosomes, derived from each parental species. The basic genetic sex in bitterlings is male (ZZ) and the derivative is female (ZW). When parental species are related, the sex phenotype of hybrids coincides with the genetic sex. However, when the parental species differ, the sex phenotype of the ZW genotype is reversed to become male by an abnormal interaction between the Z and W chromosomes. The rare appearance of females and intersexes in male-predominant hybrids might be due to complete or partial functional expression of the W chromosome.     

Stability of inactive X-chromosome in mouse embryoid body-mule cell and transformed mouse cell-mule cell heterokaryons

Cloned fibroblast cells of female mule derivation, expressing only the horse G6PD phenotype, were fused with established mouse cells, with spontaneously and virally transformed mouse cells, and with embryoid bodies from a transplantable mouse teratoma. Heterokaryons were harvested at various intervals after fusion, and tested for their G6PD activity patterns by electrophoresis on Cellogel sheets. No expression of the donkey G6PD phenotype in these heterokaryons could be detected, although hybrid G6PD bands formed between mouse and horse subunits were clearly visible. These observations imply that neither the cytoplasm of the embryoid bodies, nor of the DNA and RNA tumour virus-transformed mouse cells can induce depression of the G6PD locus on an inactive X-chromosome and provide further evidence for the genetic stability of the inactive X-chromosome in mammalian somatic cells.     

Modulation of allospecific CTL responses during pregnancy in equids: an immunological barrier to interspecies matings?

Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags. This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly, mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity. The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle barrier to reproductive success between species.