The present invention mainly relates to a method free of pharmaceutical products for improving fattening in mammals by altering the normal information existing between both uterine horns and the ovaries of domestic female mammals by means of a intrauterine device, with the object of making a hormonal alteration leading to the suppression of the estrous cycle. the absence of ovulation, and the increase in the daily weight gain particularly of cows and heifers.

Intrauterine devices (IUD) are the most efficient birth-control method in humans (World Health Organization, 1987). Their mechanism. which consists in suppressing human fertility, has been subject to controversy because of the diversity of methods found in trials undergone by animals of different species (Marston and Kelly, 1966).

As far as women and non-human primates are concerned, the IUD interferes with fertilization while the menstrual cycle remains undisturbed (Chi, 1993).

Rodent ovulation, spermatic transport and fertilization are unaltered by IUD action, however it interferes with the process of implantation by means of these three mechanisms:

• a) embryonic degeneration,
• b) acceleration of embryonic transit and ejection,
• c) alteration of the implanting uterine component or decidua (Marston and Kelly, 1996).

In rodents with two uterine horns and two independent uterine necks. such as rats and rabbits, anti-implantation effects are reached when a IUD is placed in each horn (Davis, 1972). In the case of mice, both horns are joined by a common neck. that is why the insertion of a IUD in one horn is sufficient so as to impede the embryonic implantation in both horns (Marston and Kelly. 1966).

In relation with rodents and ruminants having two independent uterine horns. contraceptive effects are reached only when a IUD is placed in each horn (Marston and Kelly, 1966, Davis 1972. Hawk et al 1973).

In the case of ruminants such as sheep and cows apart from contraceptive effects, there is an alteration of the oestrous cycle according to the distention degree caused by the IUD in the uterus (Nalbandov et al., 1955; Hansel & Wagner, 1960; Turin et al., 1966).

The insertion of a IUD in sheep interferes with the oestrous periodicity without restraining ovulation. The contraceptive effect, in this case, seems to be the result of an alteration in the spermatic transport and an early luteinization. The origin of this phenomenon is possibly related to the uterine distention after the insertion of a IUD these circumstances are manifested when the IUD is placed homolaterally with respect to the ovulating ovary.

Specialized literature suggests that the insertion of IUD in cows does not modify the oestrous intensity, but it has a contraceptive effect by means of an alteration in the spermatic transport and an early luteinization caused by uterine distention. When talking about contraceptive efficiency. size and location of lUDs seem to be key factors (Marston and Kelly, 1966; Hawk et al.), 1968).

The method consists in inserting (by means of a specially designed applicator) at least one device into the uterine cavity, particularly one in each uterine horn, at least.

According to what is experimentally known, there is a variation according to the species regarding the IUD contraceptive effect on most mammal species having mechanisms that control the effects against fertility.

Human and non-human primates have a menstrual cycle which is unaltered by lUDs and their natural ovulation cycles remain unaffected. The uterine device applied according to the present invention interferes with the oestrous period since it affects the ovarian function. The effects of this device seem to be critical for ruminants because it increases the uterine distention occurring in the uterus after it is located in one or both uterine corns. The following description relates to the effects of a copper bearing device specially designed for altering the ovarian function, reproductive capacity and weight gain of cows and heifers.

Intents of weight gain were effected in the past based on animal castration. In 1906, Dutto affirmed that Nuffer was the first one to employ surgical practices with ulterior ovary ectomy. Furthermore Dutto also reported bloody techniques carried out in the past by Charlier and Degive. Dutto also designed the ovarytome, an instrument which improved Degive's cruel technique of bonding ovaries. This technique is still being used at present in spite of the fact that not only acts in a bloody manner, but also has a variable success depending on the ovary bond position and its peritoneal revascularization.

It is known that weight gain is one of the objects of our cattle breeding However This method consisting of intrauterine devices can only be applicable to the females. Female bovine animals have what is called sexual cycle. which is central to coition. This cycle involves four phases: the oestrum period, also called mating season, which lasts 24 hours approximately and is the only time the female is willing to mate, the precedent period is called pro-oestrum and, in case coition does not result in conception, there is a following phase which is called metoestrum, afterwards a pause called diostrum period takes place, and then a new cycle starts. Specific hormones are involved so as to reach a normal and timely completion of the cycle.

Ovulation takes place during the oestrum period in which the mature ovule is released by the ovary. Once the ovulation has started. the luteuos corpus is formed in the ovary. This corpus is in charge of producing Progesterone, a hormone of important anabolic effects in charge of controlling coming cycles and pregnancy normal development. In case conception is unfruitful, the luteous corpus is disintegrated by a hormone called Prostaglandine F2, and thus a new sexual cycle starts.

The synthesis of Prostaglandine F2 alpha occurs in the uterus, more precisely in the endometrium, then it reaches the luteous corpus by means of a mechanism called uterus-ovary crosscurrent which disintegrates it before the beginning of a new cycle.

In view of the above, it should be mentioned that unless Prostaglandine F2 alpha is present, the luteous corpus will not be disintegrated and will continuously generate Progesterone for an undetermined period causing important effects:

• absence of oestrum, ovulation and conception caused by a suprabasal level of Progesterone generated by persistent luteous structures. This suprabasal level blocks the beginning of a new cycle by inhibiting the follicle hormones (unovulation)
• anabolic effect: the techa luteinization alters the techa/granular relation and the excessive substance produced by the luteinizated internal theca (hypertrophy) fails to aromatize in granulose and to turn into estrogen. Consequently, androgens are released increasing the concentration six or seven times, varying from 0.026 to 0.163 nanograms/ml. Thus, these androgens are responsible of weight gain.

More precisely, the object of the present invention comprises a method of implanting a device in each of the uterine horns, in order to block the normal function existing between the uterus and the ovaries, thus preventing Prostaglandina F2 alpha from synthesizing

Besides, the present invention relates to the device intended to be inserted in the bovine uterine cavity. This consists of an elongated main body with a head located at its upper extreme and two or more arms located at its lower extreme having an inverted "V" or "U" shape -in case the number of arms is two- and also having a lineal slit therein, the body is coiled by a copper filament

• Figure 1 is an elevational side view of the device.
• Figure 2 shows a variant embodiment of the device, and
• Figure 3 shows another variant embodiment of the device.

References 1, 2 , and 3 corresponding to the above mentioned figures stand for the head (1), the body (2) and the arms (3) of the device respectively.

Figures 4 to 6 will be described below.

It is important to bear in mind that the slit at the lower surface of each arm serves the purpose of avoiding material fatigue and causing a snapping effect to keep the arms open. The function performed by the arms is to provide force and contact with the endometrium of the uterus of the cow so as to obtain the desired results.

It should be pointed out that the device in Figure 1 is intended for cows and heifers and the devices in Figures 2 and 3, which are bigger, are intended for dairy cows having larger uterus. A pyramidal, rounded or oval head is located in the other extreme of the main body. The uterine device corresponding to the present invention is intended for influencing cow fattening rhythm and handling.

Basically, the functional characteristic of this device is to remain inside the uterus of the mammal in order to cause a persistent irritation in the endometrium which is complemented by the action of the copper (metallosys) and the distention of the walls of uterine horns. This way, the synthesis of Prostaglandine F2 would be avoided causing the animal to have a period of oestral absence, being the luteus corpus persistent, Progesterone is consequently released by this structure

This involves three basic steps:

• 1. Alteration of the normal functioning of the endometrium cell due to copper action which releases ions,
• 2. Irritation by mechanic action, and
• 3. Distention of the walls of the uterine horns.

In order to facilitate the understanding of the constitutive, functional and emplacement characteristics of the method described in the present invention, an embodiment example is described below and shown schematically in the attached sheet without specific scale, with the precise explanation regarding the non-restrictive or exclusive character of the example according to the scope of protection of the present invention, in contrast, the example is to be considered only as merely explanatory of the fundamental conception on which the invention is based.

The constitutive components of the present invention are (1) a head, (2) a body, and (3) two arms, as shown in Figures 1, 2, and 3. The head, which can be pyramidal, rounded or oval shaped, serves as a cervical expander due to the fact that this method is applied during different cycle periods and the cervix can be probably closed.. Body size ranges from 1 (one) to 4 (four) centimeters in length and from 1 (one) to 4 (four) millimeters in diameter, varying according to uterus size This body is coiled by a copper filament. Arms ranges from 3 (three) to 10 (ten) centimeters in length and from 2 (two) to 8 (eight) millimeters in diameter. There is a "spun" stainless steel bar inside the body, used as a plunger, which serves the purpose of removing the device from the inserter tube once it is located into the uterine horns. Besides two polypropylene knobs are specially designed to facilitate device handling.

Device insertion comprises the following steps:

• 1. The animal is immobilized by means of an animal trap and then the tail is raised.
• 2. The inserter tube bearing the devices therein is submerged into a receptacle containing disinfectant solution basically constituted by 10% iodine.
• 3. Vulvar lips are separated and the inserter tube is placed in the vaginal vestibule up to the cervix.
• 4. The right arm of the inserter tube is placed into the animal rectus, the cervix is strongly fixed and guided frontward by means of the thumb, index and middle fingers.
• 5. The end of the inserter tube composed by the device head is inserted into the cervix lengthwise in order to reach the uterine body.
• 6. Then one of the horns is reached and the device or devices are placed in the uterine horn bottom.
• 7. The inserter tube is again placed in the uterine body. the operator takes the other horn with his right hand and places the inserter tube in the other horn to insert the remaining device or devices therein.

Subsequently, the applicator is removed and the process ends.

MATERIALS AND METHODS

Animals

Four hundred and sixty nuliparous heifers, weighing 210 to 325 kg were subject to this study. Most were Hereford and Angus breed but some were Hereford-Angus crossbreed. The animals belonged to different farms with feeding and husbandry practices that were uniform for all animals within each farm. All animals were examined by rectus palpation for the purpose of detecting possible reproductive problems and, at least two oestroum periods were detected prior to be randomly assigned to receive the IUD (n-230) or to serve as control (n-230).

Intrauterine Device (IUD)

The device was developed with a shape and size enough to distend the uterine cavity slightly in nuliparous heifers.

The two side arms , of 2.5 mm in diameter and 3.5 cm in length, open and expand at the distal end of the main elongated body giving a maximal anchor of 3.5 cm. Two devices are loaded into an inserter tube with an outer diameter of 0.4 cm and 44.5 cm in length. Each device with its folded arms is located immediately below the second one in the tube. The folded flexible arms of each IUD remain open as they left the tube.

To study the effects of IUD on weight gain, oestrus cycle and hormonal profile on day 0-10 of the cycle (day 0=oestrum), the animals were weighed and a heparinized blood sample was obtained via jugular puncture and subsquently a IUD was inserted in each uterine horn. As shown in Figure 5. the insertion tube holds two lUDs. The same Figure shows the moment the operator inserts by plunger each device with its extremes folded. Each tube is guided through the cervix and into each uterine horn by manipulation of the cervix per rectum and the first IUD is slowly ejected by plunger into the uterine lumen. The tube is then inserted in the other horn and the second device was ejected as well. Heparinized biood is sampled again 24 and 48 ns after the procedure and 20 and 120 days later Blood is refrigerated and subject to centrifugation at 1.500 g for 10 min and aliquots of plasma are stored at -20 °C until assayed. IUD-treated and control animals were checked twice daily for oestrus and controlled at 60 and 120 days for weight gain

To study the effects of IUD on pregnancy, IUD-treated (n-200) and control (n=200) animals were naturally mated within a period of 90 days. Pregnancy was confirmed by rectal or external abdominal palpation.

Hormonal Assays

Progesterone and testosterone concentrations were determined for all plasma samples, by radioimmunoassay. Briefly, plasma aliquots of 200 ul were extracted in 10 vol of petroleum ether. The extracts were brought to dryness in air and reconstituted in 200 ul of buffer and 25 - 50 ul aliquots were assayed in duplicate. The monoclonal antiserum for progesterone assay was raised against progesterone-11-hemisuccinate-BSA and used to a final 1:40.000 dilution. The monoclonal antiserum for progesterone assay was raised against testosterone-3-(carboximethyl)-oxime-BSA and used to a final 1:650.000 dilution. Other steroids had < 10% crossreaction with these antisera. The anti-body hormone complex was precipitated with rabbit antimouse gamma globulin bound to magnetic particles. For both progesterone and testosterone. the coefficient of intrassay variation was between 5 and 10%, for the coefficient of interassay variation based on repeated measurements, in different assay runs, of aliquots from pools of plasma with high and low progesterone and testosterone concentrations, were between 7 and 15%. Recovery values after extraction were consistently about 92% for progesterone and testerone respectively.

A representative number of animals of each group was sacrified before preeding and blocks of ovaries and uterus preserved in formalin were embedded in paraffin sectioned at 8 u and stained with hematoxilin and eosin for histological evaluation.

RESULTS

The effects of IUD on body weight gain, oestrous cycle and pregnancy rate are shown in Table 1. During the first sixty days of the study, the daily gain in body weight for helfers bearing the IUDs was 16.5% higher than for the control group (p<0.05). For the period from 61 to 120 days IUD-treated heifers averaged a daily weight gain 25.5% greater than for controi animals (p<0.001). These figures represent a significant increase (p<0.05) in the rate of body weight gain for heifers bearing the lUDs between day 60 and 120. By the contrary, in such period, control heifers showed a daily weight gain similar to that observed in the first sixty days.

Daily Weight gain (gr./day)

Number of estrus in 120 days

Pregnancy rate (%) 120 days

0-60

61-120

Control

712+31a

709+23b

1055 (100)*

97

IUD

830+27c

890+19d

21 (3)*

0

*Percent of animals showing oestrous

a vs. c:<0.05, b vs d: p<0.001; c vs. d:p<0.05

All control heifers showed oestrous over the period of study, totalling 1055 cycles in 120 days. By the contrary, the insertion of lUDs in both uterine horns caused anestrus in 98% of the animals and only 5 out of 230 IUD-treated heifers totalled 21 oestrous throughout the study. Following these studies, most of control and IUD-treated heifers including those that showed some estrus, were mated naturally during a period of 120 days. All control calves become pregnant within the first 90 days of the breeding period. By the contrary, no pregnancies were detected in IUD-treated heifers until 70 days after the end of the breeding period. The effects of IUD on the variable levels of progesterone and testosterone are shown in Fig. 4. At 0 time both, control and IUD-treated animals, showed similar progesterone concentrations (control 4.2 +0.3ng/ml : IUD 3.9+0.1 ng/ml) indicating a normal luteal development at the time of IUD insertion. At this basal stage, testosterone levels were 25.2+6.2 and 22.5+12pg/ml for control and IUD groups respectively. No changes occurred, neither in progesterone nor testosterone levels, in the first 40 hs after IUD insertion On day 20, whereas control heifers were in a new luteal phase with an average progesterone concentration of 3.3 +0.8ng/ml, and unchanged testosterone levels, IUD-treated heifers showed a significant (p<0.001) reduction in progesterone to a mean concentration of 0.7+0.3 ng/ml. Simultaneously, testosterone have increased four times in these animals to a mean value of 106+170pg/ml. Or, day 120, whereas control heifers showed a wide rage in progesterone values with an average of 2.7+0.9 ng/ml, and unchanged testosterone values, the heifers bearing the lUDs had consistently low progesterone values and significantly higher testosterone levels. reaching a mean concentration of 163+23 pg/ml.

Associated histological preparations from ovaries of IUD-treated heifers are shown in Figure 6. There was no evidence of recent pending ovulation in no one of the ovaries examined. The sections revealed the presence of 2 or more cysts per ovary The cysts were surrounded by a thick wall with yellow pigment at various points within the theca cells layers. The ovaries also had many atrophic antral follicles. Some cysts and many antral follicles exhibited marked hyperthecosis with luteinization, hyperchromatism and abundant blood vessels. There was a fibrous thickening of the tunic albuginea. In some restricted areas of ovaries cortex there were a few primordial follicles.

CONCLUSIONS

The results of the present study reveal a high efficacy of IUD as a contraceptive in heifers. The lUDs exert their contraceptive function by inducing a continuous anestrous condition.

Probably, as result of the uterine distention caused by the IUD, the ovarian folliculogenesis and steroiedogenesis were modified resulting in a failure in the ovulatory process with the moderate hyperandrogenism. The increase in the circulating levels of testosterone and its well known anabolic effects and the anestrus status were the main responsible factors for the improvement in body weight gain that was observed in IUD-treated heifers.