Identification of X-chromatin and determination of its incidence in Nigerian goat breeds

J C Okonkwo, I S Omeje* and U N Egu**

Department of Agricultural and Bio-resources Engineering, Nnamdi Azikiwe University, PMB 5025 Awka, Nigeria
* Department of Animal Science and Fisheries, Delta State University, Asaba, Delta State
** Department of Animal Science and Fishery, Abia State University, PMB 7010 Umuahia, Abia State, Nigeria
jossychidiebere@yahoo.com

Abstract

The numbers of x-chromatin body (“drumstick” appendages) on the polymorph nuclear neutrophils (PMNS) in three major breeds of Nigerian goats were studied using 81 goats (27 goats per breed). Each goat breed was derived from three different locations in the country based on the areas of its preponderance. Smears from buccal cavity and PMNS from each goat were developed using standard staining techniques.

The mean values obtained per breed irrespective of sex were 1.92%, 1.65% and 1.60% for Sahel Goats (SG), Red Sokoto Goats (RSG) and West African Dwarf Goats (WADG), respectively. The mean values obtained for the bucks and does; irrespective of breed were 0.13% and 3.07%, respectively. Those for males per breed were 0.15% for SG, 0.15% for RSG, and 0.10% for WADG and for does per breed were 3.44%    for SG,   3.10% for RSG   and 2.67% for WADG. The results generally revealed that the frequency of ‘drumstick’ was statistically different (P<0.05) between Bucks and Does; Bucks were statistically the same (P<0.05) in ‘drumstick’ incidence, irrespective of breed and location, while the ‘drumstick’ incidence was statistically higher (P<0.05) in Sahelian Does, followed by RS Does and least in WAD Does. This may account for higher prolificacy frequently observed in SG, followed by RSG.  However, location exhibited an infinitesimal effect on the frequency of ‘drumstick’ within breeds, indicating that it’s incidence is purely a genetic factor.

Key words: drumstick, location, sex, and source within breed

Introduction

Typical female X-chromatin body in the polymorph nuclear neutrophils (PMNS) of cattle, buffaloes and goats appears as a drumstick attachment to a thread similar to that found in human PMNS (Kalva 1981; Bhatia and Shanker 1993, 1992a). The X-chromatin can be found anywhere on the nucleus jutting slightly from the tip of the main nucleus to form the sex chromatin bodies. It has a round or oval head with an average size of 1.77µ x 1.0µ in the goat (Singh 1987; Bhatia and Shanker 1982b). However, structural abnormalities of the X-chromosome have been reported to affect the size and shape of the X-chromatin (Monre 1996; Ford and Evans 1977). Drumsticks tend to be larger than usual in females carrying one normal and one large X chromosomes (xX). On the other hand, if a normal X-chromosome is paired with a partially deleted chromosome (x_x), the appendage then becomes comparatively smaller (Bhatia and Shanker 1993, 1992b, 1992a, 1980).

Barr bodies result from the inactive, or heterochromatinization of all but one of the X chromosome (Satbir et al 2006). That is, a mammalian female with two X chromosome will have one of these chromosomes inactivated. Lyon (1972) reported that the dosage relationship between the sex chromosome (in female) and autosomes are the same as in somatic tissue of male. Researchers have reviewed various models on how this inactivation occurs. In general, inactivation of additional X-chromosome occurs in a random fashion. Satbir et al (2006), Sanhya (1994) and Hartwell et al (2000) claim that quantitatively, the effect of X-chromosome inactivation can be detected by measuring the amount of protein produced by a sex-linked gene in individuals carrying a number of chromosomes since female heterozygousity for sex-linked recessive genes does not show mosiacism. According to Lyon (1972) many individuals with extra X-chromosomes are seriously affected with some deleterious traits ranging from physical deformation to mental retardation. However, individuals without any X-chromosome are never found, probably because of in-viability (Satbir et al 2006; Fecheimer and Harper 1980).

The inactive chromosome (Xb) does not express majority of its genes, unlike the active (Xa) chromosome. This is due to silencing of the Xb by the repressive heterochromatic, which cover the Xb genes/DNA and prevent the expression of most of its genes (Russell 1980; Sanhya 1994; Monre 1996). When compared with Xa, the Xb has high level of DNA methylation, low level of histone acetylation, low level of histon H3 lysine-4 methylation and high level of histone H3 lysine-9 methylation, all of which are associated with gene silencing. In addition, a histone vilant protein called macro H2A is exclusively found in Xb (Satbir et al 2006; Standford 1982; Hartwell et al 2000).   It has been shown that only fractions of the inactivated X-chromosome are actually inactive, and certain areas crucial to the development remain active (Russell 1980; Hartwell et al 2000). This X-chromosome is usually replicating, highly coiled, condensed, and may be visualized microscopically as clear dense mass against the inner part of the nuclear membrane. This dense mass is referred to as the X-chromatin or simply the barr body after Murry Barr who first discovered the structure (Barr and Betram 1949; Moore and Barr 1954). These researchers have advocated the presence and absence of the barr body in the PMNS may be used as a cyto-diagnostic tool for determination of gender, and prolificacy in livestock. The researchers maintained that males are X-chromatin negative while the females are positive. Some further reported the percentage range of 2.5-3.5 for females. The purpose of this study therefore, is to establish the drumstick profiles of the Nigerian goats and asses the effects of breed, sex and geographical distance within breed on this parameter.

Materials and methods 

Sources of the livestock and experimental Site

Three major Nigerian indigenous goat breeds were randomly selected from nine different locations in the country based on areas of its predominance. A total nine goats (comprising three males and six females) were used per location. The goat breeds and locations chosen are: Sahel goat (Maiduguri, Potiskum, and Gumel); Red Sokoto goat (Sokoto, Katsina and Gusau), and West African Dwarf goat (Umuahia, Ughelli, and Akure). The latitude of Nigeria falls within the tropical zone but the climatic conditions are not entirely tropical in nature, but vary in most parts of the country; in the north the climatic condition is arid and to the south there is an equatorial type of climate. Maiduguri, Potiskum and Gumel lies within latitude and longitude of 12°0'N and 13°20'E, 11°39'N and 11°02'E, and 12°39'N and 09°22'E, respectively. Sokoto, Katsina and Gusau fall within latitude and longitude 13°2'N and 5°16'E, 13°00'N and 07°32'E, and 12°12'N and 06°40'E, respectively while Umuahia, Ughelli, and Akure are within latitude and longitude 05°10'N and 7°19'E, 5°50'N and 5°40'E, and 07°15'N and 05°05'E, respectively.

The goats were sourced and housed at the Teaching and Research Farm of the Department of Animal Science and Fisheries, Faculty of Agriculture, Delta State University, Asaba Campus, Asaba, Delta State. Delta State falls within the humid tropics of Nigeria, and Asaba precisely lies between longitudes 6⁰E and 8⁰E, and between latitude 4⁰N and 10⁰N. It has a moderate climate with a very high temperature during the dry season and average rainfall during the rainy season. Asaba has the mean annual temperature and precipitation of 28⁰C and 1,450mm, respectively (NIMET 2006).

Design of the experiment

The experiment was conducted under a 2 x 3 factorial in completely randomized design (CRD) to test the effects of sex, breed, and their interactions on the drumstick incidence of the Nigerian indigenous goats. Sex was tested on two levels with unequal replicates of twenty-seven bucks and fifty-four does; while there were three breeds with twenty-seven goats for each breed. In addition, effect of source of goat within each breed on this parameter was tested using one-way classification.

The statistical model used:

Y_{ijk =}µ+B_i +S_j + (BS)_ij + e_ijkl

Where:

Y_{ijk -}is the observed drumstick incidence arising as a result of:

µ -the population mean; 

B_i – the effect of the breed, _i= 1, --, 3

S_{j –} the effect of _jth sex of the animal, _j=1, 2

(BS)_ij- is the interaction between breed and sex, and 

e_ijk –is the error term associated with the observations.

Assumptions; error term is independently, identically and normally distributed, with zero mean and constant variance, that is, IIND (0, σ²).

Analytical procedure                       

Determination of ‘Drumsticks’ frequency from the buccal mucosa

The goats were not given access to feed two hours prior to sample collection, but were allowed access to good volume of water in order to cut down the bacteria load. Using spatula, inside of the goat’s cheek was scraped. The first two samples collected were discarded by wiping the spatula on a clean towel. This was done to cut down on the bacterial counts of the sample. With the same tool, third sample was collected from the same area, and used to make a smear on a clean glass slide. Two smears were prepared from each goat. The slides were immediately placed in a fixative containing 75% methyl alcohol and 25% acetic acid for one hour. The slides with fixed buccal mucosa smears were immersed in a staining jar containing 1%creyl violet acetate for 5minutes. Thereafter they were rinsed by dipping quickly into three changes (separate jars) of 95% ethanol. Again, they were dipped into two changes of xylol and placed in a fresh xylol for 2minutes. The slides were air-dried overnight. The slides were examined using transmitted light (x400 magnification). 200 nuclei (100 per slide) were counted. Nuclei that are broken, folded, or excessively granular were not counted. Areas heavily contaminated by debris or bacteria were avoided. A nucleus was regarded as positive when it contains an X chromatin body that is clearly defined, dark staining, and touching the nuclear membrane.

Determination of ‘Drumsticks’ incidence from the Polymorph Nuclear Neutrophils (PMNS)

The blood sample collected was placed in EDTA containing tube, manually shaken and centrifuged to hasten the separation of the blood constituents into the white blood cells, red blood cells, and the plasma. Then, the procedure adopted for buccal mucosa was employed except that Leishman’s stain was used instead of creyl violet acetate.

Statistical analysis

All the data collected were subjected to analysis of variance (ANOVA) appropriate for a 2x3 factorial in a CRD to test the effects of breed, sex and their interactions on the measured parameter. However, percentage values were transformed into Arcsine angles prior to analysis. The differences between means were separated using Duncan’s New Multiple Range Test (DNMRT).

Results  

Drumstick incidence in the buccal cavity

The mean values of the drumstick incidence as determined from the buccal cavity for each breed, and with respect to sex are given in Table 1.

Significant differences (P<0.05) were observed between the breeds. Furthermore, when the drumstick incidence was considered for each sex within breed, it was observed that the males exhibited no significant differences between them in the breeds.

Sahel does were significantly higher (P<0.05) than the does of other breeds. The Red Sokoto does have significantly higher (P<0.05) drumstick incidence than the West African dwarf does. The values ranging from 0.15 ± 0.12 to 0.22 ± 0.11 were observed for the bucks across the board, while the range from 2.75 ±  0.01 to 3.55 ±  0.01 was obtained for the does, respectively.

Source within breed effects

The mean values of drumstick incidence observed in each location in each breed are presented in Table 2.

The results revealed that there is no significant difference (P>0.05) in drumstick incidence among goats derived from various geographical locations. This fact holds even when you consider the goats irrespective of sex.

Drumstick incidence in polymorphonuclear cells (PMNS)

The frequencies of drumsticks obtained from the polymorphous neutrophilic nuclei for each breed, and for each sex are presented in Table 3. 

The results followed the same pattern as when measured from the buccal cavity. Again, no significant differences (P>0.05) were observed between the bucks from different breeds, but when the goats were considered irrespective of sex, differences (P<0.05) were observed. The SG does were significantly higher (P<0.05) than other breeds, followed by RSG does.

Source within breed effects

The mean values of drumsticks observed from the PMNS for each location within each breed are presented in Table 4.

There were no significant differences (P>0.05) between various locations for the same breed on the incidence of drumstick in the PMNS. So, none of the breeds is affected by location on the incidence of drumstick, irrespective of sex.
 

Discussion

The disparity in drumsticks incidence with respect to sex observed is in agreement with what have been obtained in the world goat breeds (Omeje et al 1994; Bhatia and Shanker 1992b; 1992a, 1983b, 1980). The negligible levels of drumstick occurrences recorded in the bucks in this study are a credit to the Nigerian goat breeds, and fall within the range of 0.0-0.5% documented in the literature (Bhatia and Shanker 1983b, 1983a, 1980; Omeje and Berepubo 1996). This confirmed the reproductive soundness of the male goats. The drumsticks frequency ranges found in the Nigerian does lie within the range of 2.59-3.6% reported by Bhatia and Shanker (1983b). This suggests that Nigerian does are inherently prolific and have high potential for the development of parents and grandparents stocks as reported by Omeje (2004). Again, breed differences observed in drumsticks incidences in the female sub-group, indicates that the Sahelian does have higher prolificacy, followed by the Red Sokoto does. This might be responsible for the multiply births (triplets, quadruplets and so on) often encountered in the SG breed, and twinning (sometimes triplets) in the RSGs (Aliyu 1990; FAO 1985). This disparity calls for serious attention into the Nigerian major goat breeds, and development of a breeding programme which will combine the varied inherent potentials in these breeds. A host of literature reports abound that the SG has poor meat quality and meat yield; the RSG has excellent meat yield, meat quality, and the skins are highly prized even in international markets, and the WADG is resistant to trypanosomiasis and tolerates harsh environmental conditions (Oyenuga 1976; Matthewman 1977; Mason 1984; Ademosun 1994).

Furthermore, though the mean values of the breeds were considered excellent, few does in the groups recorded very low levels of drumsticks incidences, which is indicative of several abnormalities which, probably, might include chimerism or mixoploidy. Bhatia and Shanker (1980) reported that female goats with less than 1% drumsticks appendages per 200 PMNS will display impaired fertility. Chimerism interferes with the reproductive efficiencies of does because of the effect on their reproductive hormones and enzymes (Harmerton 1971; Long and Williams 1980; Omeje and Berepubo 1996). They further stated that phenotypic females with features of ovarian dysgenesis and low chromatin positive counts would be suggestive of mixoploidy (45x/ 46xx). Also, few males with high drumsticks frequencies were encountered in all the breeds, and equally exhibit differential fertility. Cytodiagnostic evaluation of the drumsticks incidence is a prerequisite for selecting breeding does and bucks.

No significant source within breed effect was observed in drumsticks incidences. This implies that drumstick incidence is purely a genetic factor distinct for each breed, and expresses itself regardless of environment.  

Conclusions 

• The drumstick incidence may be determined from either the buccal cavity or the polymorph nuclear cells (PMNS) of goats with equal efficacy and accuracy. The percentage drumsticks in Nigeria goats is sex specific, with the bucks having as low as 0.10-0.22% and the does 2.75-3.55%. Breed has no effect on the frequency of drumstick in male goats, but exhibited highly significant effect on the female sub-group. The does of the Sahelian breed have the highest percentage of the drumstick, followed by the Red Sokoto does, while the does of the West African Dwarf  have the least values. The percentages of the drumsticks for does of the three breeds of the Nigerian goats are in consonance with the documentations for other world breeds of goat. Consequently, the Nigerian goats have some inherent potential for high fertility and productivities. 

  
  

• The multiple births and high milk yield characteristics of the SG breed are inherent abilities that this breed is noted for, and which may not be unrelated to the high incidence of drumsticks observed. The average performance of the RSGs in these traits- multiple births and milk yield is a reflection of the percentage of drumsticks in their PMNS. Also, poor productivity of the WADGs in multiple births and milk yield compared to the other breeds could be due to lower percentage of the drumstick in the breed. Generally, drumstick incidence is purely a genetic factor, and geneticists should study the trait with a view to identify, isolate and use the gene in establishment of parent and grandparent stocks ideal for milk yield and multiple births. Furthermore farmers should establish a cyto diagnostics Laboratories to enable them cull inferior animals early in life.

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Received 28 January 2010; Accepted 13 November 2010; Published 9 December 2010

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